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Normothermic machine perfusion (NMP) is a clinical strategy to reduce renal ischemia-reperfusion injury (IRI). Optimal NMP should restore metabolism and minimize IRI induced inflammatory responses. Microdialysis was used to evaluate renal metabolism. This study aimed to assess the effect of complement inhibition on NMP induced inflammatory responses. Twenty-two pig kidneys underwent 18 h of static cold storage (SCS) followed by 4 h of NMP using a closed-circuit system. Kidneys were randomized to receive a C5-inhibitor or placebo during SCS and NMP. Perfusion resulted in rapidly stabilized renal flow, low renal resistance, and urine production. During SCS, tissue microdialysate levels of glucose and pyruvate decreased significantly, whereas glycerol increased (p < 0.001). In the first hour of NMP, glucose and pyruvate increased while glycerol decreased (p < 0.001). After 4 h, all metabolites had returned to baseline. Inflammatory markers C3a, soluble C5b-9, TNF, IL-6, IL-1β, IL-8, and IL-10 increased significantly during NMP in perfusate and kidney tissue. C5-inhibition significantly decreased perfusate and urine soluble C5b-9 ( p < 0.001; p = 0.002, respectively), and tissue IL-1β ( p = 0.049), but did not alter other inflammatory markers. Microdialysis can accurately monitor the effect of NMP on renal metabolism. Closed-circuit NMP induces inflammation, which appeared partly complement-mediated. Targeting additional immune inhibitors should be the next step., Competing Interests: TM is a consultant for UCB Pharma. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 de Boer, Sokolova, Jager, Schjalm, Weiss, Liavåg, Maassen, van Goor, Thorgersen, Pettersen, Christiansen, Ludviksen, Jespersen, Mollnes, Leuvenink and Pischke.)

The advent of recycling antibodies, leveraging pH-dependent antigen binding and optimized FcRn interaction, has advanced the field of antibody therapies, enabling extended durability and reduced dosages. Eculizumab (Soliris ® ) demonstrated the efficacy of C5 inhibitors for paroxysmal nocturnal hemoglobinuria (PNH), while its derivative, ravulizumab (Ultomiris ® ), recognized as a recycling antibody, extended the dosing intervals. However, limitations including intravenous administration and inefficacy in patients with the R885H single-nucleotide polymorphism (SNP) in C5 could necessitate alternative solutions. Crovalimab (PiaSky ® ), a next-generation recycling antibody, overcomes these challenges with innovative charge engineering, achieving the enhanced cellular uptake of C5-crovalimab complexes and targeting a unique C5 epitope, allowing for efficacy regardless of the R885H SNP. This study highlights crovalimab's distinctive molecular features, showing its eliminated binding to Fcγ receptors and C1q, alongside its optimized antigen binding characteristics. The impact of charge engineering was reconfirmed in mice, demonstrating faster C5 clearance than recycling antibodies. Notably, in the maintenance dosing regimen, crovalimab neutralizes approximately seven C5 molecules per antibody on average. Furthermore, its design also reduces the viscosity to facilitate high-concentration formulations suitable for subcutaneous delivery. Consequently, crovalimab offers a four-weekly subcutaneous injection regimen for PNH, marking a substantial improvement in treatment convenience and potentially transforming patients' quality of life.

Emerging evidence indicates that activation of complement system leading to the formation of the membrane attack complex (MAC) plays a detrimental role in COVID-19. However, their pathogenic roles have never been experimentally investigated before. We used three knock out mice strains (1. C3 -/- ; 2. C7 -/- ; and 3. Cd59ab -/- ) to evaluate the role of complement in severe COVID-19 pathogenesis. C3 deficient mice lack a key common component of all three complement activation pathways and are unable to generate C3 and C5 convertases. C7 deficient mice lack a complement protein needed for MAC formation. Cd59ab deficient mice lack an important inhibitor of MAC formation. We also used anti-C5 antibody to block and evaluate the therapeutic potential of inhibiting MAC formation. We demonstrate that inhibition of complement activation (in C3 -/- ) and MAC formation (in C3 -/- . C7 -/- , and anti-C5 antibody) attenuates severe COVID-19; whereas enhancement of MAC formation (Cd59ab -/- ) accelerates severe COVID-19. The degree of MAC but not C3 deposits in the lungs of C3 -/- , C7 -/- mice, and Cd59ab -/- mice as compared to their control mice is associated with the attenuation or acceleration of SARS-CoV-2-induced disease. Further, the lack of terminal complement activation for the formation of MAC in C7 deficient mice protects endothelial dysfunction, which is associated with the attenuation of diseases and pathologic changes. Our results demonstrated the causative effect of MAC in severe COVID-19 and indicate a potential avenue for modulating the complement system and MAC formation in the treatment of severe COVID-19., (© 2024. The Author(s).)

Background: Respiratory failure can be a severe complication after polytrauma. Extensive systemic inflammation due to surgical interventions, as well as exacerbated post-traumatic immune responses influence the occurrence and progression of respiratory failure. This study investigated the effect of different surgical treatment modalities as well as combined inhibition of the complement component C5 and the toll-like receptor molecule CD14 (C5/CD14 inhibition) on the pulmonary microRNA (miRNA) signature after polytrauma, using a translational porcine polytrauma model., Methods: After induction of general anesthesia, animals were subjected to polytrauma, consisting of blunt chest trauma, bilateral femur fractures, hemorrhagic shock, and liver laceration. One sham group (n=6) and three treatment groups were defined; Early Total Care (ETC, n=8), Damage Control Orthopedics (DCO, n=8), and ETC + C5/CD14 inhibition (n=4). Animals were medically and operatively stabilized, and treated in an ICU setting for 72 h. Lung tissue was sampled, miRNAs were isolated, transcribed, and pooled for qPCR array analyses, followed by validation in the individual animal population. Lastly, mRNA target prediction was performed followed by functional enrichment analyses., Results: The miRNA arrays identified six significantly deregulated miRNAs in lung tissue. In the DCO group, miR-129, miR-192, miR-194, miR-382, and miR-503 were significantly upregulated compared to the ETC group. The miRNA expression profiles in the ETC + C5/CD14 inhibition group approximated those of the DCO group. Bioinformatic analysis revealed mRNA targets and signaling pathways related to alveolar edema, pulmonary fibrosis, inflammation response, and leukocytes recruitment. Collectively, the DCO group, as well as the ETC + C5/CD14 inhibition group, revealed more anti-inflammatory and regenerative miRNA expression profiles., Conclusion: This study showed that reduced surgical invasiveness and combining ETC with C5/CD14 inhibition can contribute to the reduction of pulmonary complications., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Zhou, Groven, Horst, Mert, Greven, Mollnes, Huber-Lang, van Griensven, Hildebrand and Balmayor.)

The rising prevalence of fungal infections is a significant and growing public health threat, and this risk is further underscored by our incomplete understanding of why organs like the kidney are so susceptible to systemic candidiasis. To combat the high mortality of such infections, we urgently need to advance our understanding of fungal pathogenesis and how it articulates with human immune response. Now, a recent landmark study has illuminated a crucial role of the complement system in the response to candidiasis and determined the stepwise local response of phagocytes within the kidney during infection. These fundamental discoveries provide crucial insights that can be leveraged to improve the care and outcome for patients with fungal infections.

Myopia is regarded as a worldwide epidemic ocular disease, has been proved related to inflammation. CD55, also known as decay-accelerating factor (DAF) can modulate the activation of complement through inhibiting the formation of complement 3 convertase and its dysregulation is involved in various inflammatory diseases. To investigate the association between CD55 and myopia, and to test whether CD55 can inhibit myopia development by suppressing inflammation in the eye, we use three different animal models including monocular form-deprivation myopia, myopia induced by TNF-α administration and allergic conjunctivitis animal model to reveal the CD55 in myopia development. The tears of thirty-eight participants with different spherical equivalents were collected and CD55 in the tears were also analyzed. Complement 3 and complement 5 levels increased while CD55 levels decreased in allergic conjunctivitis and myopic eyes. After anti-inflammatory drugs administration, CD55 expression was increased in monocular form-deprivation myopia model. We also found inflammatory cytokines TGF-β, IL-6, TNF-α, and IL-1β may enhance complement 3 and complement 5 activation while CD55 level was suppressed contrary. Moreover, lower CD55 levels were found in the tears of patients with myopia with decreased diopter values. Finally, CD55-Fc administration on the eyelids can inhibit the elongation of axial length and change of refractive error. CD55-Fc application also suppress myopia development subsequent to complement 3 and complement 5 reduction and can lower myopia-specific (MMP-2 and TGF-β) cytokine expression in TNF-α induced myopia animal model. This suggests that CD55 can inhibit myopia development by suppression of complement activation and eventual down-regulation of inflammation., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Aims: Vascular calcification (VC) predicts the morbidity and mortality in cardiovascular diseases. Vascular smooth muscle cells (VSMCs) osteogenic transdifferentiation is the crucial pathological basis for VC. To date, the molecular pathogenesis is still largely unclear. Notably, C5a-C5aR1 contributes to the development of cardiovascular diseases, and its closely related to physiological bone mineralization which is similar to VSMCs osteogenic transdifferentiation. However, the role and underlying mechanisms of C5a-C5aR1 in VC remain unexplored., Methods and Results: A cross-sectional clinical study was utilized to examine the association between C5a and VC. Chronic kidney diseases mice and calcifying VSMCs models were established to investigate the effect of C5a-C5aR1 in VC, evaluated by changes in calcium deposition and osteogenic markers. The cross-sectional study identified that high level of C5a was associated with increased risk of VC. C5a dose-responsively accelerated VSMCs osteogenic transdifferentiation accompanying with increased the expression of C5aR1. Meanwhile, the antagonists of C5aR1, PMX 53, reduced calcium deposition, and osteogenic transdifferentiation both in vivo and in vitro. Mechanistically, C5a-C5aR1 induced endoplasmic reticulum (ER) stress and then activated PERK-eIF2α-ATF4 pathway to accelerated VSMCs osteogenic transdifferentiation. In addition, cAMP-response element-binding protein 3-like 1 (CREB3L1) was a key downstream mediator of PERK-eIF2α-ATF4 pathway which accelerated VSMCs osteogenic transdifferentiation by promoting the expression of COL1α1., Conclusions: High level of C5a was associated with increased risk of VC, and it accelerated VC by activating the receptor C5aR1. PERK-eIF2α-ATF4-CREB3L1 pathway of ER stress was activated by C5a-C5aR1, hence promoting VSMCs osteogenic transdifferentiation. Targeting C5 or C5aR1 may be an appealing therapeutic target for VC., Competing Interests: Conflict of interest: None declared., (© The Author(s) 2023. Published by Oxford University Press on behalf of the European Society of Cardiology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Systemic candidiasis is a common, high-mortality, nosocomial fungal infection. Unexpectedly, it has emerged as a complication of anti-complement C5-targeted monoclonal antibody treatment, indicating a critical niche for C5 in antifungal immunity. We identified transcription of complement system genes as the top biological pathway induced in candidemic patients and as predictive of candidemia. Mechanistically, C5a-C5aR1 promoted fungal clearance and host survival in a mouse model of systemic candidiasis by stimulating phagocyte effector function and ERK- and AKT-dependent survival in infected tissues. C5ar1 ablation rewired macrophage metabolism downstream of mTOR, promoting their apoptosis and enhancing mortality through kidney injury. Besides hepatocyte-derived C5, local C5 produced intrinsically by phagocytes provided a key substrate for antifungal protection. Lower serum C5a concentrations or a C5 polymorphism that decreases leukocyte C5 expression correlated independently with poor patient outcomes. Thus, local, phagocyte-derived C5 production licenses phagocyte antimicrobial function and confers innate protection during systemic fungal infection., Competing Interests: Declaration of interests The authors declare no competing interests., (Published by Elsevier Inc.)

African Trypanosomes have developed elaborate mechanisms to escape the adaptive immune response, but little is known about complement evasion particularly at the early stage of infection. Here we show that ISG65 of the human-infective parasite Trypanosoma brucei gambiense is a receptor for human complement factor C3 and its activation fragments and that it takes over a role in selective inhibition of the alternative pathway C5 convertase and thus abrogation of the terminal pathway. No deposition of C4b, as part of the classical and lectin pathway convertases, was detected on trypanosomes. We present the cryo-electron microscopy (EM) structures of native C3 and C3b in complex with ISG65 which reveal a set of modes of complement interaction. Based on these findings, we propose a model for receptor-ligand interactions as they occur at the plasma membrane of blood-stage trypanosomes and may facilitate innate immune escape of the parasite., (© 2023. The Author(s).)

Introduction: Complement-based drug discovery is undergoing a renaissance, empowered by new advances in structural biology, complement biology and drug development. Certain components of the complement pathway, particularly C1q and C3, have been extensively studied in the context of neurodegenerative disease, and established as key therapeutic targets. C5 also has huge therapeutic potential in this arena, with its druggability clearly demonstrated by the success of C5-inhibitor eculizumab., Areas Covered: We will discuss the evidence supporting C5 as a target in neurodegenerative disease, along with the current progress in developing different classes of C5 inhibitors and the gaps in knowledge that will help progress in the field., Expert Opinion: Validation of C5 as a therapeutic target for neurodegenerative disease would represent a major step forward for complement therapeutics research and has the potential to furnish disease-modifying drugs for millions of patients suffering worldwide. Key hurdles that need to be overcome for this to be achieved are understanding how C5a and C5b should be targeted to bring therapeutic benefit and demonstrating the ability to target C5 without creating vulnerability to infection in patients. This requires greater biological elucidation of its precise role in disease pathogenesis, supported by better chemical/biological tools.

Over the last decade, perspectives on the complement system in the context of cancer have shifted, with complement proteins now implicated in many of the hallmarks of cancer. Systemically, the generation of complement anaphylatoxin C5a, the most potent inflammatory mediator of the cascade, occurs following convertase-mediated cleavage of complement component C5. In a recent manuscript, Ding et al., propose that in colorectal cancer cells, C5 cleavage can occur intracellularly and in a convertase-independent manner, identifying cathepsin D as an enzyme capable of cleaving C5 into C5a [1]. Intracellular C5a is functional and promotes β-catenin stabilisation via the assembly of a KCTD5/cullin3/Roc-1 complex. Importantly, the blockade of C5aR1 prevents tumorigenesis. This study adds to a growing body of evidence indicating that complement proteins, previously thought to primarily have extracellular or membrane-bound functions, also have important intracellular roles., (© 2023. The Author(s).)

Over a century after the discovery of the complement system, the first complement therapeutic was approved for the treatment of paroxysmal nocturnal hemoglobinuria (PNH). It was a long-acting monoclonal antibody (aka 5G1-1, 5G1.1, h5G1.1, and now known as eculizumab) that targets C5, specifically preventing the generation of C5a, a potent anaphylatoxin, and C5b, the first step in the eventual formation of membrane attack complex. The enormous clinical and financial success of eculizumab across four diseases (PNH, atypical hemolytic uremic syndrome (aHUS), myasthenia gravis (MG), and anti-aquaporin-4 (AQP4) antibody-positive neuromyelitis optica spectrum disorder (NMOSD)) has fueled a surge in complement therapeutics, especially targeting diseases with an underlying complement pathophysiology for which anti-C5 therapy is ineffective. Intensive research has also uncovered challenges that arise from C5 blockade. For example, PNH patients can still face extravascular hemolysis or pharmacodynamic breakthrough of complement suppression during complement-amplifying conditions. These "side" effects of a stoichiometric inhibitor like eculizumab were unexpected and are incompatible with some of our accepted knowledge of the complement cascade. And they are not unique to C5 inhibition. Indeed, "exceptions" to the rules of complement biology abound and have led to unprecedented and surprising insights. In this review, we will describe initial, present and future aspects of protein inhibitors of the complement cascade, highlighting unexpected findings that are redefining some of the mechanistic foundations upon which the complement cascade is organized., (© 2022 The Authors. Immunological Reviews published by John Wiley & Sons Ltd.)

Bisphenol AF (BPAF) is one of the substitutes for bisphenol A (BPA), which has endocrine-disrupting, reproductive and neurological toxicity. BPAF has frequently been detected in the aquatic environment, which has been a long-term threat to the health of aquatic organisms. In this study, female marine medaka (Oryzias melastigma) were exposed to 6.7 μg/L, 73.4 μg/L, and 367.0 μg/L BPAF for 120 d. The effects of BPAF on behavior, growth, liver and ovarian histology, gene transcriptional profiles, and reproduction of marine medaka were determined. The results showed that with the increase of BPAF concentration, the swimming speed of female marine medaka showed an increasing trend and then decreasing trend. BPAF (367.0 μg/L) significantly increased body weight and condition factors in females. BPAF (73.4 μg/L and 367.0 μg/L) significantly delayed oocyte maturation. Exposure to 367.0 μg/L BPAF showed an increasing trend in the transcript levels of lipid synthesis and transport-related genes such as fatty acid synthase (fasn), sterol regulatory element binding protein (srebf), diacylglycerol acyltransferase (dgat), solute carrier family 27 member 4 (slc27a4), fatty acid-binding protein (fabp), and peroxisome proliferator-activated receptor gamma (pparγ) in the liver. In addition, 6.7 μg/L BPAF significantly down-regulated the expression levels of antioxidant-related genes [superoxide dismutase (sod), glutathione peroxidase (gpx), and catalase (cat)], and complement system-related genes [complement component 5 (c5), complement component 7a (c7a), mannan-binding lectin serine peptidase 1 (masp1), and tumor necrosis factor (tnf)] were significantly up-regulated in the 73.4 and 367.0 μg/L groups, which implies the effect of BPAF on the immune system in the liver. In the hypothalamic-pituitary-ovarian axis (HPG) results, the transcription levels of estrogen receptor α (erα), estrogen receptor β (erβ), androgen receptor (arα), gonadotropin-releasing hormone 2 (gnrh2), cytochrome P450 19b (cyp19b), aromatase (cyp19a), and luteinizing hormone receptor (lhr) in the brain and ovary, and vitellogenin (vtg) and choriogenin (chg) in the liver of 367.0 μg/L BPAF group showed a downward trend. In addition, exposure to 367.0 μg/L BPAF for 120 d inhibited the spawning behavior of marine medaka. Our results showed that long-term BPAF treatment influenced growth (body weight and condition factors), lipid metabolism, and ovarian maturation, and significantly altered the immune response and the transcriptional expression levels of HPG axis-related genes., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Invasive aspergillosis (IA) is a life-threatening fungal infection for immunocompromised hosts. It is, therefore, necessary to understand the immune pathways that control this infection. Although the primary infection site is the lungs, aspergillosis can disseminate to other organs through unknown mechanisms. Herein we have examined the in vivo role of various complement pathways as well as the complement receptors C3aR and C5aR1 during experimental systemic infection by Aspergillus fumigatus , the main species responsible for IA. We show that C3 knockout (C3 -/- ) mice are highly susceptible to systemic infection of A. fumigatus . Intriguingly, C4 -/- and factor B (FB) -/- mice showed susceptibility similar to the wild-type mice, suggesting that either the complement pathways display functional redundancy during infection (i.e., one pathway compensates for the loss of the other), or complement is activated non-canonically by A. fumigatus protease. Our in vitro study substantiates the presence of C3 and C5 cleaving proteases in A. fumigatus . Examination of the importance of the terminal complement pathway employing C5 -/- and C5aR1 -/- mice reveals that it plays a vital role in the conidial clearance. This, in part, is due to the increased conidial uptake by phagocytes. Together, our data suggest that the complement deficiency enhances the susceptibility to systemic infection by A. fumigatus ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Shende, Wong, Meitei, Lal, Madan, Aimanianda, Pal and Sahu.)

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.

Neuromyelitis optica (NMO) is an inflammatory disease that resembles MS in the relapsing clinical course of optic neuritis and myelitis. Two decades of studies have revealed that autoantibodies, reactive to the water channel protein aquaporin 4 (AQP4) are detected in the core group of patients. These autoantibodies play a crucial role in the inflammatory pathology of NMO, involving proinflammatory cytokines, chemokines, and various inflammatory cells such as Th17 cells. Anti-AQP4 antibody-positive NMO differs fundamentally from MS, particularly in the responsiveness to therapies and the neuropathology accompanying destruction of astrocytes. Research into the immunological mechanism has led to the identification of possible targets of therapy, including complement pathway and interleukin-6 (IL-6) receptor signaling. Recent randomized controlled clinical trials have shown the remarkable efficacy of antibodies specific for complement C5, IL-6 receptor, and CD19 + B cells in prevention of NMO spectrum disorder relapses, although no such effects were found in anti-AQP4 antibody-negative patients. These results imply that anti-AQP4 antibody is a biomarker predicting the efficacy of therapies, and indicate the future direction towards "precision medicine.", (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Rationale: Doublecortin-like kinase 1 (DCLK1) is a serine/threonine kinase that selectively marks cancer stem-like cells (CSCs) and promotes malignant progression in colorectal cancer (CRC). However, the exact molecular mechanism by which DCLK1 drives the aggressive phenotype of cancer cells is incompletely determined. Methods: Here, we performed comprehensive genomics and proteomics analyses to identify binding proteins of DCLK1 and discovered X-ray repair cross-complementing 5 (XRCC5). Thus, we explored the biological role and downstream events of the DCLK1/XRCC5 axis in human CRC cells and CRC mouse models. Results: The results of comprehensive bioinformatics analyses suggested that DCLK1-driven CRC aggressiveness is linked to inflammation. Mechanistically, DCLK1 bound and phosphorylated XRCC5, which in turn transcriptionally activated cyclooxygenase-2 expression and enhanced prostaglandin E 2 production; these events collectively generated the inflammatory tumor microenvironment and enhanced the aggressive behavior of CRC cells. Consistent with the discovered mechanism, inhibition of DCLK1 kinase activity strongly impaired the tumor seeding and growth capabilities in CRC mouse models. Conclusion: Our study illuminates a novel mechanism that mediates the pro-inflammatory function of CSCs in driving the aggressive phenotype of CRC, broadening the biological function of DCLK1 in CRC., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Background: Crovalimab is a humanized monoclonal antibody targeting human complement C5. Patients switching from eculizumab to crovalimab are expected to form drug-target-drug complexes (DTDCs), since these antibodies each bind to a different epitope on complement C5. An analytical method to evaluate the size distribution of these DTDCs was developed and validated. Methods: Human serum samples were separated by size-exclusion chromatography (SEC) into eight fractions, and the concentration of crovalimab in each fraction was measured by ELISA. We evaluated SEC, ELISA and the combination of both methods (SEC-ELISA). Results: Predetermined validation acceptance criteria were met. Conclusion: The DTDC assay method was successfully validated. It enables us to evaluate the impact of DTDCs on clinical outcomes.

Exposure of the lens to UVB can lead to oxidative stress, which would result in age-related cataract (ARC) formation. In this study, we investigate the regulatory mechanism of tripartite motif containing 25 (TRIM25) in ARC. The protein level of TRIM25 was elevated in ARC specimens and UVB-exposed SRA01/04 cells. Bioinformatic analysis indicated that X-ray repair cross complementing 5 (XRCC5) might interact with TRIM25, and the interaction was validated via immunoprecipitation. TRIM25 interacted with XRCC5 and ubiquitinated it for degradation. Further studies showed that XRCC5 overexpression notably repressed UVB-induced apoptosis, while XRCC5 knockdown promoted apoptosis. Of note, ubiquitination of XRCC5 mediated by TRIM25 overexpression facilitated apoptosis. Attenuation of XRCC5 ubiquitination by mutant with substitution of lysine residues with arginine residues rescued its anti-apoptosis effect. Moreover, we observed that TRIM25-mediated XRCC5 degradation was reversed by proteasome inhibitor MG-132 or lysosome inhibitor 3-MA. In conclusion, TRIM25 mediates ubiquitination of XRCC5 to regulate the function and degradation of XRCC5, suggesting that interventions targeting TRIM25 might be a promising therapeutic strategy for ARC., (Copyright © 2022 Elsevier Inc. All rights reserved.)

It is still a big puzzle how ovarian cancer cells and the tumor microenvironment (TME) attract lymphocytes infiltration for facilitating metastasis, a leading cause of death from gynecological malignancies. Using genome-wide LncRNA microarray assay, here we report that a LncRNA associated with ovarian cancer metastasis (LncOVM) is highly correlated with poor prognosis and survival. LncOVM interacts with and stabilizes PPIP5K2 by suppressing ubiquitinated degradation to promote complement C5 secretion from ovarian cancer cells. The TME-enriched complement C5 attracts myeloid-derived suppressor cells (MDSCs) infiltration in TME to facilitate metastasis. Knockdown of LncOVM or PPIP5K2 inhibits tumor progression in xenograft models. Application of C5aR antibody or inhibitor (CCX168) inhibits MDSC recruitment and restores the suppression of tumorigenesis and metastasis in vivo . Our study reveals that suppression of ovarian cancer metastasis can be achieved by targeting MDSC infiltration in TME through disrupting LncOVM-PPIP5K2-complement axis, providing an option for treating ovarian cancer patients., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Introduction: Both increased activity of the complement system (CS) and the role of the pituitary hormone prolactin (PRL) are implicated in osteoarthritis (OA) pathogenesis. Besides, Cathepsin D (CatD) activity is increased in the context of OA and can exert not only proteolytic but also non-proteolytic effects on cells. For the first time, possible crosstalk between two separate humoral systems: the CS and the PRL hormone systems in chondrocytes are examined together., Methods: Primary human articular chondrocytes (hAC) were stimulated with complement protein C5 (10 µg /mL), PRL (25 ng/mL), CatD (100 ng/mL), or anaphylatoxin C5a (25 ng/mL) for 24 h or 72 h, while unstimulated cells served as controls. In addition, co-stimulations of C5 or PRL with CatD were carried out under the same conditions. The influence of the stimulants on cell viability, cell proliferation, and metabolic activity of hAC, the chondrosarcoma cell line OUMS-27, and endothelial cells of the human umbilical cord vein (HUVEC) was investigated. Gene expression analysis of C5a receptor (C5aR1), C5, complement regulatory protein CD59, PRL, PRL receptor (PRLR), CatD, and matrix metal-loproteinases (MMP)-13 were performed using real-time PCR. Also, collagen type (Col) I, Col II, C5aR1, CD59, and PRL were detected on protein level using immunofluorescence labeling., Results: The stimulation of the hAC showed no significant impairment of the cell viability. C5, C5a, and PRL induced cell growth in OUMS-27 and HUVEC, but not in chondrocytes. CatD, as well as C5, significantly reduced the gene expression of CatD, C5aR1, C5, and CD59. PRLR gene expression was likewise impaired by C5, C5a, and PRL+CatD stimulation. On the protein level, CatD, as well as C5a, decreased Col II as well as C5aR1 synthesis., Conclusions: The significant suppression of the C5 gene expression under the influence of PRL+CatD and that of CD59 via PRL+/-CatD and conversely a suppression of the PRLR gene expression via C5 alone or C5a stimulation indicates an interrelation between the two mentioned systems. In addition, CatD and C5, in contrast to PRL, directly mediate possible negative feedback of their own gene expression.

Glycolipids are now known to be rapidly converted to mediators for inflammatory reactions or to signaling molecules that control inflammatory events in the nervous system. The present study aimed to explore whether disturbed glycolipids metabolism in the nervous system is present in patients with a neuroinflammatory disorder, encephalo-myelo-radiculo-neuropathy (EMRN), because most EMRN patients have been reported to exhibit autoantibodies against neutral glycolipids. Although molecular pathogenesis of this disorder remains unknown, we tried to search the immunochemical abnormalities in this disorder. ELISA for activated peripheral C5 complement and mass spectrometry analysis of cerebrospinal fluid clearly disclosed a significant upregulation of active C5 complement, C5a levels in sera as well as a significant accumulation of species-specific ceramides but not sphingomyelin in cerebrospinal fluid from EMRN patients. Furthermore, we confirmed the occurrence of anti-neutral glycolipids antibodies in all EMRN patients. Thus, the present study might indicate the pathophysiology of this disorder is the dysregulation of glycolipids metabolism and abnormal production of autoantibodies against neutral glycolipids resulting in the abnormal complement activation, although molecular basis for these sphingolipids dysregulation and the occurrence of autoantibodies against glycolipids remains to be elucidated at present. The present study implicates a new therapeutic strategy employing anti-ceramide and/or anti-complement therapy for this disorder., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The complement system plays a role in the formation of sub-retinal pigment epithelial (RPE) deposits in early stages of age-related macular degeneration (AMD). But the specific mechanisms that connect complement activation and deposit formation in AMD patients are unknown, which limits the development of efficient therapies to reduce or stop disease progression. We have previously demonstrated that C3 blockage prevents the formation of sub-RPE deposits in a mouse model of EFEMP1-associated macular degeneration. In this study, we have used double mutant Efemp1 R345W/R345W :C5 -/- mice to investigate the role of C5 in the formation of sub-RPE deposits in vivo and in vitro. The data revealed that the genetic ablation of C5 does not eliminate the formation of sub-RPE deposits. Contrarily, the absence of C5 in RPE cultures promotes complement dysregulation that results in increased activation of C3, which likely contributes to deposit formation even in the absence of EFEMP1-R345W mutant protein. The results also suggest that genetic ablation of C5 alters the extracellular matrix turnover through an effect on matrix metalloproteinases in RPE cell cultures. These results confirm that C3 rather than C5 could be an effective therapeutic target to treat early AMD.

Background: Osteoarthritis is currently one of the most common chronic diseases. As life expectancy increases, its prevalence and incidence are expected to rise. At present, more and more evidences prove the correlation between the complement system and osteoarthritis (OA). This study aims to investigate complement C5's influence on the effect of MK801 on osteoarthritis synovial fibroblasts (OA-SFs)., Methods: We used IL-1b to induce OA-SFs derived from mice to obtain OA-SFs. And we performed RT-PCR and Western Blot assays to evaluate the expression levels of associated mRNA and protein. The alteration of MAC expression on OA-SFs cell membrane was evaluated by immunofluorescence assay. The expression of related inflammatory factors of OA-SFs was evaluated by ELISA experiment., Results: MK801 could significantly inhibit the expression of osteoarthritis (OA) marker factors, such as: membrane attack complex (MAC), tumor necrosis factor-α (TNF-α) and matrix metalloproteinase-13 (MMP13). Meanwhile, MK801 can significantly inhibit the expression of complement C5 (C5) in OA-SFs. Immunofluorescence assay showed that MAC expression on OA-SFs cell membrane was significantly inhibited by MK801. The nucleo-plasmic separation experiment demonstrated that MK801 could significantly inhibit the activation of Nuclear factor-κB (NF-κB) signaling pathway in OA-SFs. Futhermore, koncking down the expression of C5 reversed the inhibition MK801 on the expression of OA-SFs inflammatory factors., Conclusions: These results illustrated two points: first, MK801 inhibited the generation of MAC and the release of inflammation factors in OA-SFs through C5; second: MK801 inhibited the activation of NF-κB signaling pathway in OA-SFs., (Copyright © 2021. Published by Elsevier Ltd.)

Complement is an important innate immune defence machinery. Once dysregulated, it is often linked to pathogenesis of diverse autoimmune diseases. Artesunate (ART) is a well-known anti-malarial compound. Recently, ART has been highlighted by its potential therapeutic effects on certain complement-related autoimmune diseases. However, the underlying mechanisms are hitherto unknown. In the present study, we found that ART mediated complement interception as validated by analysis of complement haemolytic assay. In cell-based setup using dying Jurkat cells, ART-mediated complement interception was also confirmed. Further, we newly established an ELISA system selectively allowing complement activation via the classical pathway, the lectin pathway and the alternative pathway, respectively. ELISA analysis revealed that ART dose-dependently inhibited C4 activation, C3 activation and terminal complement complex assembly via the effector pathways. ART was found to blockade C1q, C3 and C5 with a lesser extent to properdin. The interaction of ART with C1q was determined to be mediated via C1q globular head region. FACS analysis using ART-conjugated mesoporous silica particles revealed that ART specifically bound the key therapeutic targets of C1q, C3 and C5 on microparticles. In conclusion, we for the first time report the anti-complement bioactivities of ART and suggest a potential therapeutic benefit of ART in the complement-related human diseases., (Copyright © 2021 The Author(s). Published by Elsevier Masson SAS.. All rights reserved.)

Ravulizumab (Ultomiris ® ), a humanized monoclonal antibody that inhibits complement protein C5, is indicated for the treatment of atypical haemolytic uraemic syndrome (aHUS) in several countries, including the USA and those of the EU. Ravulizumab has been re-engineered from eculizumab to extend its terminal elimination half-life, resulting in a more convenient maintenance dosage regimen of once every 4-8 weeks compared with once every 2-3 weeks for eculizumab. In single-arm phase 3 trials, ravulizumab resolved thrombotic microangiopathy in 54% and 78% of treatment-naïve adult and paediatric patients with aHUS, respectively, within 26 weeks. Ravulizumab was also effective in patients with postpartum aHUS and paediatric patients who responded to eculizumab and later switched to ravulizumab. Ravulizumab was generally well tolerated, with no unexpected safety events. The most common treatment-related adverse events with ravulizumab in treatment-naïve patients include headache, diarrhoea and vomiting. With its convenient once every 4-8 weeks maintenance regimen, ravulizumab is an important treatment option for aHUS in adult and paediatric patients.

Periodontitis is the inflammatory destruction of the tooth-surrounding and -supporting tissue, resulting at worst in tooth loss. Another locally aggressive disease of the oral cavity is tooth resorption (TR). This is associated with the destruction of the dental mineralized tissue. However, the underlying pathomechanisms remain unknown. The complement system, as well as mast cells (MCs), are known to be involved in osteoclastogenesis and bone loss. The complement factors C3 and C5 were previously identified as key players in periodontal disease. Therefore, we hypothesize that complement factors and MCs might play a role in alveolar bone and tooth resorption. To investigate this, we used the cat as a model because of the naturally occurring high prevalence of both these disorders in this species. Teeth, gingiva samples and serum were collected from domestic cats, which had an appointment for dental treatment under anesthesia, as well as from healthy cats. Histological analyses, immunohistochemical staining and the CH-50 and AH-50 assays revealed increased numbers of osteoclasts and MCs, as well as complement activity in cats with TR. Calcifications score in the gingiva was highest in animals that suffer from TR. This indicates that MCs and the complement system are involved in the destruction of the mineralized tissue in this condition.

Ravulizumab every 8 weeks showed non-inferiority to eculizumab every 2 weeks in a 26-week, phase 3, randomized controlled trial in adults with paroxysmal nocturnal hemoglobinuria (PNH) who were clinically stable on eculizumab (NCT03056040). We report results from the first 26 weeks of the extension period in which patients continued ravulizumab (n = 96) or switched from eculizumab to ravulizumab (n = 95). At week 52, mean (SD) lactate dehydrogenase levels increased 8.8% (29%) with ravulizumab-ravulizumab and 5.8% (27%) with eculizumab-ravulizumab from primary evaluation period baseline. During the extension period, four patients (ravulizumab-ravulizumab, n = 3; eculizumab-ravulizumab, n = 1) experienced breakthrough hemolysis, but none associated with serum free C5 ≥ 0.5 μg/mL. Mean Functional Assessment of Chronic Illness Therapy (FACIT)-Fatigue scores remained stable through week 52. During the extension period, proportions of patients avoiding transfusion remained stable (ravulizumab-ravulizumab, 86.5%; eculizumab-ravulizumab, 83.2%); 81.2% and 81.1%, respectively, had stabilized hemoglobin. All patients maintained serum free C5 levels < 0.5 μg/mL. Adverse events were generally similar between groups, and rates were lower in the extension period. Adults with PNH on stable eculizumab therapy who received ravulizumab over 52 weeks experienced durable efficacy, with consistent efficacy in patients who received eculizumab during the primary evaluation period and then switched to ravulizumab. Ravulizumab was well tolerated., (© 2020 The Authors. European Journal of Haematology published by John Wiley & Sons Ltd.)

The homeostasis of tissues in a chronic disease is an essential function of the alternative pathway (AP) of the complement system (CS). However, if not controlled, it may also be detrimental to healthy cells with a consequent aggravation of symptoms. The protoporphyria (PP) is a rare chronic disease that causes phototoxicity in visible light with local skin pain and general malaise. In order to establish if there is a systemic involvement of the CS during sun exposure, we designed a non-invasive method with a serum collection in winter and summer from 19 PP and 13 controls to detect the levels of CS protein: Properdin, Factor H (FH), and C5. Moreover, the global radiation data were collected from the regional agency of environmental protection (ARPA). The results show growing values for every protein in patients with PP, compared to control, in both seasons, in particular in summer compared to winter. To reinforce the evidence, we have estimated the personal exposure of patients based on the global radiation data. The main factors of the AP increased over the season, confirming the involvement of the AP in relation to light exposure. The systemic response could justify the general malaise of patients after long light exposure and can be exploited to elucidate new therapeutic approaches., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer AM declared a shared affiliation with the authors SF and IM to the handling editor at the time of review., (Copyright © 2021 Granata, Duca, Brancaleoni, Fustinoni, De Luca, Motta, Graziadei and Di Pierro.)

Cross-linking mass spectrometry has developed into an important method to study protein structures and interactions. The in-solution cross-linking workflows involve time and sample consuming steps and do not provide sensible solutions for differentiating cross-links obtained from co-occurring protein oligomers, complexes, or conformers. Here we developed a cross-linking workflow combining blue native PAGE with in-gel cross-linking mass spectrometry (IGX-MS). This workflow circumvents steps, such as buffer exchange and cross-linker concentration optimization. Additionally, IGX-MS enables the parallel analysis of co-occurring protein complexes using only small amounts of sample. Another benefit of IGX-MS, demonstrated by experiments on GroEL and purified bovine heart mitochondria, is the substantial reduction of undesired over-length cross-links compared to in-solution cross-linking. We next used IGX-MS to investigate the complement components C5, C6, and their hetero-dimeric C5b6 complex. The obtained cross-links were used to generate a refined structural model of the complement component C6, resembling C6 in its inactivated state. This finding shows that IGX-MS can provide new insights into the initial stages of the terminal complement pathway., (© 2021 The Authors. Published under the terms of the CC BY 4.0 license.)

Bovines have evolved a subset of antibodies with ultra-long heavy chain complementarity determining regions that harbour cysteine-rich knob domains. To produce high-affinity peptides, we previously isolated autonomous 3-6 kDa knob domains from bovine antibodies. Here, we show that binding of four knob domain peptides elicits a range of effects on the clinically validated drug target complement C5. Allosteric mechanisms predominated, with one peptide selectively inhibiting C5 cleavage by the alternative pathway C5 convertase, revealing a targetable mechanistic difference between the classical and alternative pathway C5 convertases. Taking a hybrid biophysical approach, we present C5-knob domain co-crystal structures and, by solution methods, observed allosteric effects propagating >50 Å from the binding sites. This study expands the therapeutic scope of C5, presents new inhibitors, and introduces knob domains as new, low molecular weight antibody fragments, with therapeutic potential., Competing Interests: AM, ZA, JB, MS, BH, VO, JS, VE, AL employee of UCB and may hold shares and/or stock options, ML, MG, SC, SR, PN, DS, Jv No competing interests declared, TM T.E.M is a Board member of Ra Pharmaceuticals, Inc, CD Reviewing editor, eLife, (© 2021, Macpherson et al.)

Complement hyperactivation, angiopathic thrombosis and protein-losing enteropathy (CHAPLE disease) is a lethal disease caused by genetic loss of the complement regulatory protein CD55, leading to overactivation of complement and innate immunity together with immunodeficiency due to immunoglobulin wasting in the intestine. We report in vivo human data accumulated using the complement C5 inhibitor eculizumab for the medical treatment of patients with CHAPLE disease. We observed cessation of gastrointestinal pathology together with restoration of normal immunity and metabolism. We found that patients rapidly renormalized immunoglobulin concentrations and other serum proteins as revealed by aptamer profiling, re-established a healthy gut microbiome, discontinued immunoglobulin replacement and other treatments and exhibited catch-up growth. Thus, we show that blockade of C5 by eculizumab effectively re-establishes regulation of the innate immune complement system to substantially reduce the pathophysiological manifestations of CD55 deficiency in humans.

Nonmelanoma skin cancer such as cutaneous squamous cell carcinoma (cSCC) is the most common form of cancer and can occur as a consequence of DNA damage to the epithelium by UVR or chemical carcinogens. There is growing evidence that the complement system is involved in cancer immune surveillance; however, its role in cSCC remains unclear. Here, we show that complement genes are expressed in tissue from patients with cSCC, and C3 activation fragments are present in cSCC biopsies, indicating complement activation. Using a range of complement-deficient mice in a two-stage mouse model of chemically-induced cSCC, where a subclinical dose of 7,12-dimethylbenz[a]anthracene causes oncogenic mutations in epithelial cells and 12-O-tetradecanoylphorbol-13-acetate promotes the outgrowth of these cells, we found that C3-deficient mice displayed a significantly reduced tumor burden, whereas an opposite phenotype was observed in mice lacking C5aR1, C5aR2, and C3a receptor. In addition, in mice unable to form the membrane attack complex, the tumor progression was unaltered. C3 deficiency did not affect the cancer response to 7,12-dimethylbenz[a]anthracene treatment alone but reduced the epidermal hyperplasia during 12-O-tetradecanoylphorbol-13-acetate-induced inflammation. Collectively, these data indicate that C3 drives tumorigenesis during chronic skin inflammation, independently of the downstream generation of C5a or membrane attack complex., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

SKY59 or RO7112689 is a humanized monoclonal antibody against complement protein C5 with pH-dependent C5-binding and neonatal Fc receptor-mediated recycling capabilities, which result in long-lasting neutralization of C5. We developed and validated a novel total drug assay for quantification of target-binding competent SKY59 in the presence of endogenous C5 in cynomolgus monkey plasma. The target-binding competent SKY59 was determined after complex formation by the addition of recombinant monkey C5 using goat anti-human IgG-heavy chain monkey-adsorbed polyclonal antibody as a capture antibody and rabbit anti-C5 monoclonal antibody (mAb) non-competing with SKY59 for detection. The total SKY59 assay was shown to be accurate and precise over the range of 0.05-3.2 μg/mL as well as be tolerant to more than 400 μg/mL of C5 (~ 3000-fold molar excess of target). We also developed and validated a total C5 assay, confirmed selectivity and parallelism, and verified the utility of recombinant monkey C5 for the total C5 assay as well as the total SKY59 assay. Furthermore, we used these validated methods to measure SKY59 and C5 concentrations in cynomolgus monkey plasma samples in a toxicology study. This total drug assay can be applied not only to other antibody therapeutics against shed/soluble targets when a non-competing reagent mAb is available but also for clinical studies when a reagent mAb specific for engineered Fc region on a therapeutic mAb is available.

Diabetic kidney disease (DKD) is a severe DM complication. While complement C5 up-regulation and gut dysbiosis are found in T2DM, their roles in DKD are unclear. Here, we investigated the effect of C5 on the gut microbiota during DKD development. Renal C5a/C5a receptor (C5aR) expression changes were measured in T2DM patients and db/db mice. Db/db mice were treated with a C5aR antagonist (C5aRA), and renal function, gut microbiota and renal genome changes were analysed. The effects of C5a and short-chain fatty acids (SCFAs) on the signal transducer and activator of transcription 3 (STAT3) pathway were examined in vitro. C5a was up-regulated in glomerular endothelial cells (GECs) of T2DM patients and db/db mice. Although glucose and lipid metabolism were unchanged, C5aR blockade alleviated renal dysfunction, ECM deposition, macrophage infiltration and proinflammatory factor expression in db/db mice. C5aRA partly reversed the declines in gut microbiota diversity and abundance and gut SCFA levels in db/db mice. C5aRA down-regulated the expression of many immune response-related genes, such as STAT3, in db/db mouse kidneys. C5aRA and SCFAs suppressed C5a-induced STAT3 activation in human renal glomerular endothelial cells (HRGECs). Based on our results, C5 hyperactivation promotes DKD by activating STAT3 in GECs and impairing the gut-kidney axis, suggesting that this hyperactivation is a potential target for the treatment of DKD., (© 2020 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Background: Hepatic ischemia/reperfusion injury (IRI) is a serious complication in liver surgeries, including transplantation. Complement activation seems to be closely involved in hepatic IRI; however, no complement-targeted intervention has been clinically applied. We investigated the therapeutic potential of Complement 5 (C5)-targeted regulation in hepatic IRI., Methods: C5-knockout (B10D2/oSn) and their corresponding wild-type mice (WT, B10D2/nSn) were exposed to 90-minute partial (70%) hepatic ischemia/reperfusion with either anti-mouse-C5 monoclonal antibody (BB5.1) or corresponding control immunoglobulin administration 30 minutes before ischemia. C5a receptor 1 antagonist was also given to WT to identify which cascade, C5a or C5b-9, is dominant., Results: C5-knockout and anti-C5-Ab administration to WT both significantly reduced serum transaminase release and histopathological damages from 2 hours after reperfusion. This improvement was characterized by significantly reduced CD41+ platelet aggregation, maintained F4/80+ cells, and decreased high-mobility group box 1 release. After 6 hours of reperfusion, the infiltration of CD11+ and Ly6-G+ cells, cytokine/chemokine expression, single-stranded DNA+ cells, and cleaved caspase-3 expression were all significantly alleviated by anti-C5-Ab. C5a receptor 1 antagonist was as effective as anti-C5-Ab for reducing transaminases., Conclusions: Anti-C5 antibody significantly ameliorated hepatic IRI, predominantly via the C5a-mediated cascade, not only by inhibiting platelet aggregation during the early phase but also by attenuating the activation of infiltrating macrophages/neutrophils and hepatocyte apoptosis in the late phase of reperfusion. Given its efficacy, clinical availability, and controllability, C5-targeted intervention may provide a novel therapeutic strategy against hepatic IRI.

The implication of complement in multiple diseases over the last 20 years has fuelled interest in developing anti-complement drugs. To date, the focus has been on C5; blocking cleavage of C5 prevents formation of two pro-inflammatory activities, C5a anaphylatoxin and membrane attack complex. The concept of C5 blockade to inhibit inflammation dates back 30 years to the description of BB5.1, an anti-C5 blocking monoclonal antibody raised in C5-deficient mice. This antibody proved an invaluable tool to demonstrate complement involvement in mouse disease models and catalysed enthusiasm for anti-complement drug development, culminating in the anti-human C5 monoclonal antibody eculizumab, the most successful anti-complement drug to date, already in clinical use for several rare diseases. Despite its key role in providing proof-of-concept for C5 blockade, the mechanism of BB5.1 inhibition remains poorly understood. Here, we characterized BB5.1 cross-species inhibition, C5 binding affinity and chain specificity. BB5.1 efficiently inhibited C5 in mouse serum but not in human or other rodent sera; it prevented C5 cleavage and C5a generation. BB5.1 bound the C5 α-chain with high affinity and slow off-rate. BB5.1 complementarity-determining regions were obtained and docking algorithms were used to predict the likely binding interface on mouse C5., (© 2020 The Authors. Immunology published by John Wiley & Sons Ltd.)

BACKGROUND Patients receiving immunosuppressive therapies might be more susceptible to COVID-19. Conversely, an exaggerated inflammatory response to the SARS-CoV-2 infection might be blunted by certain forms of immunosuppression, which could be protective. Indeed, there are data from animal models demonstrating that complement may be a part of the pathophysiology of coronavirus infections. There is also evidence from an autopsy series demonstrating complement deposition in the lungs of patients with COVID-19. This raises the question of whether patients on anti-complement therapy could be protected from COVID-19. CASE REPORT Case 1 is a 39-year-old woman with an approximately 20-year history of paroxysmal nocturnal hemoglobinuria (PNH), who had recently been switched from treatment with eculizumab to ravulizumab prior to SARS-CoV-2 infection. Case 2 is a 54-year-old woman with a cadaveric renal transplant for lupus nephritis, complicated by thrombotic microangiopathy, who was maintained on eculizumab, which she started several months before she developed the SARS-CoV-2 infection. Case 3 is a 60-year-old woman with a 14-year history of PNH, who had been treated with eculizumab since 2012, and was diagnosed with COVID-19 at the time of her scheduled infusion. All 3 patients had a relatively mild course of COVID-19. CONCLUSIONS We see no evidence of increased susceptibility to SARS-CoV-2 in these patients on anti-complement therapy, which might actually have accounted for the mild course of infection. The effect of anti-complement therapy on COVID-19 disease needs to be determined in clinical trials.

Previous studies showed that complement activation is associated with poor functional outcome after aneurysmal subarachnoid hemorrhage (SAH). We investigated whether complement activation is underlying brain injury after aneurysmal SAH (n = 7) and if it is an appropriate treatment target. We investigated complement expression in brain tissue of aneurysmal SAH patients (n = 930) and studied the role of common genetic variants in C3 and C5 genes in outcome. We analyzed plasma levels (n = 229) to identify the functionality of a single nucleotide polymorphism (SNP) associated with outcome. The time course of C5a levels was measured in plasma (n = 31) and CSF (n = 10). In an SAH mouse model, we studied the extent of microglia activation and cell death in wild-type mice, mice lacking the C5a receptor, and in mice treated with C5-specific antibodies (n = 15 per group). Brain sections from aneurysmal SAH patients showed increased presence of complement components C1q and C3/C3b/iC3B compared to controls. The complement component 5 (C5) SNP correlated with C5a plasma levels and poor disease outcome. Serial measurements in CSF revealed that C5a was > 1400-fold increased 1 day after aneurysmal SAH and then gradually decreased. C5a in plasma was 2-fold increased at days 3-10 after aneurysmal SAH. In the SAH mouse model, we observed a ≈ 40% reduction in both microglia activation and cell death in mice lacking the C5a receptor, and in mice treated with C5-specific antibodies. These data show that C5 contributes to brain injury after experimental SAH, and support further study of C5-specific antibodies as novel treatment option to reduce brain injury and improve prognosis after aneurysmal SAH.

An important effector function of the human complement system is to directly kill Gram-negative bacteria via Membrane Attack Complex (MAC) pores. MAC pores are assembled when surface-bound convertase enzymes convert C5 into C5b, which together with C6, C7, C8 and multiple copies of C9 forms a transmembrane pore that damages the bacterial cell envelope. Recently, we found that bacterial killing by MAC pores requires local conversion of C5 by surface-bound convertases. In this study we aimed to understand why local assembly of MAC pores is essential for bacterial killing. Here, we show that rapid interaction of C7 with C5b6 is required to form bactericidal MAC pores on Escherichia coli. Binding experiments with fluorescently labelled C6 show that C7 prevents release of C5b6 from the bacterial surface. Moreover, trypsin shaving experiments and atomic force microscopy revealed that this rapid interaction between C7 and C5b6 is crucial to efficiently anchor C5b-7 to the bacterial cell envelope and form complete MAC pores. Using complement-resistant clinical E. coli strains, we show that bacterial pathogens can prevent complement-dependent killing by interfering with the anchoring of C5b-7. While C5 convertase assembly was unaffected, these resistant strains blocked efficient anchoring of C5b-7 and thus prevented stable insertion of MAC pores into the bacterial cell envelope. Altogether, these findings provide basic molecular insights into how bactericidal MAC pores are assembled and how bacteria evade MAC-dependent killing., Competing Interests: The authors have declared that no competing interests exist.

Complement activation as a driver of pathology in myasthenia gravis (MG) has been appreciated for decades. The terminal complement component [membrane attack complex (MAC)] is found at the neuromuscular junctions of patients with MG. Animals with experimental autoimmune MG are dependent predominantly on an active complement system to develop weakness. Mice deficient in intrinsic complement regulatory proteins demonstrate a significant increase in the destruction of the neuromuscular junction. As subtypes of MG have been better defined, it has been appreciated that acetylcholine receptor antibody-positive disease is driven by complement activation. Preclinical assessments have confirmed that complement inhibition would be a viable therapeutic approach. Eculizumab, an antibody directed toward the C5 component of complement, was demonstrated to be effective in a Phase 3 trial with subsequent approval by the Federal Drug Administration of the United States and other worldwide regulatory agencies for its use in acetylcholine receptor antibody-positive MG. Second- and third-generation complement inhibitors are in development and approaching pivotal efficacy evaluations. This review will summarize the history and present the state of knowledge of this new therapeutic modality., (Copyright © 2020 Albazli, Kaminski and Howard.)

The terminal complement protein (C5) inhibitor eculizumab (Soliris ® ) is the first agent to be specifically approved in the EU, USA, Canada and Japan for the treatment of neuromyelitis optica spectrum disorder (NMOSD) in adults who are aquaporin-4 water channel autoantibody (AQP4-IgG) seropositive and (in the EU only) for those with a relapsing course of disease. In the phase III PREVENT trial, eculizumab significantly reduced the risk of adjudicated relapse relative to placebo in patients with AQP4-IgG-seropositive NMOSD, approximately a quarter of whom did not receive concomitant immunosuppressive therapies. The beneficial effect of eculizumab was seen across all patient subgroups analysed and was accompanied by improvements in neurological and functional disability assessments, as well as generic health-related quality of life measures; it was sustained through 4 years of treatment, according to combined data from the PREVENT trial and an interim analysis of its ongoing open-label extension study. The safety profile of eculizumab in AQP4-IgG-seropositive NMOSD was consistent with that seen for the drug in other approved indications. Thus, eculizumab provides an effective, generally well tolerated and approved treatment option for this rare, disabling and potentially life-threatening condition.

Congenital diaphragmatic hernia (CDH) is a congenital anomaly characterized by a defect in the diaphragm. Despite the recent improvements in its treatment, CDH is associated with a high rate of neonatal mortality, which is often related to pulmonary hypoplasia (PH) as well as pulmonary hypertension. A better understanding of the underlying pathological mechanisms of PH in CDH could help establish a new treatment to improve its prognosis. In this study, we investigated serum biological profiles in neonates with CDH. For comprehensive investigation, umbilical cord serum samples were collected from isolated CDH cases (n = 4) and matched healthy controls (n = 4). Samples were analyzed using liquid chromatography-tandem mass spectrometry. A total of 697 proteins were detected; of them, 98 were identified as differentially expressed proteins. Among these differentially expressed proteins, complement C1q subcomponent showed the largest fold change, followed by complement C5. In the pathway enrichment analysis, the complement and coagulation cascades expressed the most significant enrichment ( p = 2.4 × 10 -26 ). Thus, the complement pathway might play some role in the pathophysiology of CDH., Competing Interests: The authors declare that they have no conflict of interest to disclose regarding this manuscript.

The complement system is a critical component of both innate and adaptive immune responses. It has both protective and pathogenic roles in viral infections. There are no studies regarding the role of complement system in Chandipura virus (CHPV) infection. The current study has investigated the role of complement pathways in the in vitro neutralization of CHPV in Vero E6 cells. Using normal human serum (NHS), heat-inactivated serum (HIS), human serum deficient of complement factor, respective reconstituted serum, assays like in vitro neutralization, real-time PCR, and flow cytometry-based tissue culture-based limited dose assay (TC-LDA) were carried out for assessing the activation of different complement pathways. NHS from 9/10 donors showed complement dependent neutralization, reduction in viral load and decrease in percentage of CHPV-positive cells compared to their HIS counterparts. EGTA or EDTA pretreatment experiments indicated that CHPV neutralization proceeds through the alternative pathway of the complement activation. Our data showed a strong dependence on C3 for the in vitro neutralization of CHPV. Disparity in CHPV neutralization levels between factor B-deficient and reconstituted sera could be attributed to amplification loop/"tick-over" mechanism. Assays using C3, C5, and C8 deficient sera indicated that complement-mediated CHPV neutralization and suppression of CHPV infectivity are primarily through C3 and C5, and not dependent on downstream complement factor C8. With no specific anti-viral treatment/vaccine against Chandipura, the current data, elucidating role of human complement system in the neutralization of CHPV, may help in designing effective therapeutics.

We recently showed that TLR8 is critical for the detection of Gram-positive bacteria by human monocytes. Here, we hypothesized that TLR8 and complement together regulate antibacterial responses in human blood. Anticoagulated blood was treated with selective inhibitors of TLR8 and/or complement C5, and then challenged with live Streptococcus agalactiae (Group B streptococcus, GBS), Staphylococcus aureus, or Escherichia coli. Cytokine production, plasma membrane permeability, bacterial survival, phagocytosis, and activation of coagulation was examined. GBS and S. aureus, but not E. coli, triggered TLR8-dependent production of IL-12p70, IL-1β, TNF, and IL-6 in fresh human whole blood. In purified polymorphonuclear neutrophils (PMN), GBS and S. aureus induced IL-8 release in part via TLR8, whereas PMN plasma membrane leakage and extracellular DNA levels increased independently of TLR8. TLR8 was more important than C5 for bacteria-induced production of IL-12p70, IL-1β, and TNF in blood, whereas IL-8 release was more C5 dependent. Both TLR8 and C5 induced IL-6 release and activation of prothrombin cleavage, and here their combined effects were additive. Blocking of C5 or C5aR1 attenuated phagocytosis and increased the extracellular growth of GBS in blood, whereas TLR8 inhibition neither reduced phagocytosis nor intracellular killing of GBS and S. aureus. In conclusion, TLR8 is more important than C5 for production of IL-12p70, IL-1β, and TNF upon GBS and S. aureus infection in blood, whereas C5 is central for IL-8 release and phagocytosis. Both TLR8 and C5 mediate IL-6 release and activation of coagulation during challenge with Gram-positive bacteria in blood., (© 2020 The Authors. Journal of Leukocyte Biology published by Wiley Periodicals, Inc. on behalf of Society for Leukocyte Biology.)

Complement is a key first line innate host defense system in the blood of vertebrates. Upon activation, this powerful defense mechanism can elicit inflammatory responses, lyse non-self-cells, or mark them for opsonophagocytic removal. Blood-feeding arthropods thus require the ability to block host complement activation in the bloodmeal to prevent undesired cell or tissue damage during feeding. The soft tick Ornithodoros moubata produces a complement inhibitory protein, OmCI. This protein binds to a mammalian complement protein C5 and blocks further activation of complement cascades, which results in the prevention of complement-mediated bacterial killing through membrane attack complex. Interestingly, the amino acids involved in OmCI binding are highly conserved among mammalian and avian C5, but the ability of this protein to inhibit the complement from birds remains unclear. Here we demonstrated that OmCI is capable of preventing quail complement-mediated erythrocyte lysis, inhibiting the capability of this animal's complement to eliminate a serum-sensitive Lyme disease bacterial strain. We also found that the ability of OmCI to inhibit quail complement-mediated killing of Lyme disease bacteria can be extended to different domestic and wild birds. Our results illustrate the utility of OmCI to block bird complement. These results provide the foundation for further use of this protein as a tool to study the molecular basis of avian complement and pathogen evasion to such a defense mechanism., Competing Interests: Declaration of Competing Interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2019 Elsevier GmbH. All rights reserved.)

Complement component 5 (C5) is an essential factor of the defensive complement system in all vertebrates. We report the characterization of C5 cDNA and protein from Atlantic salmon (Salmo salar), a teleost fish species of high importance in aquaculture. The C5 cDNA cloned from liver is 5079 nucleotides long, whose translation product has a molecular weight of 190 kDa, with the classical β-α orientation and motifs/sites for β-α cleavage ( 678 RPKR 681 ) and cleavage by C5 convertases (R 758 ). Mass spectrometric analysis show a single N-linked, biantennary, complex glycan at N 1125 . Moreover, the N-linked glycan displays an unusual modification in the form of acetylated sialic acid residues. Three anti-C5 antisera produced in mice using purified C5 worked in immunohistochemical analyses of formalin fixed liver tissue. The purification method, whereby inactive and activated (C5b) forms were isolated, opens for interesting studies on the complement function in fish, including possible connection to stress, disease and glycosylation., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)