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Lack of complement factor C1q of the classical pathway results in severely impaired primary antibody responses. This is a paradox because antibodies, especially IgM, are the most efficient activators of the classical pathway and very little specific IgM will be present at priming. A possible explanation would be that natural IgM, binding with low affinity to the antigen, may suffice to activate complement. In support of this, mice lacking secretory IgM have an impaired antibody response, which can be rescued by transfer of non-immune IgM. Moreover, passive administration of specific IgM together with antigen enhances the antibody response in a complement-dependent fashion. To test the idea, we have used a knock-in mouse strain (C mu 13) carrying a point mutation in the IgM heavy chain, rendering the IgM unable to activate complement. Mutant mice backcrossed to BALB/c or C57BL/6 background were primed and boosted with a low dose of sheep red blood cells. Confirming earlier data, no impairment in early, primary IgM- or IgG-responses were seen in either of the C mu 13 strains. However, in one of the mutant strains, late primary IgG responses were impaired. A more pronounced effect was observed after boost, when the IgG response, the number of germinal center B cells and antibody secreting cells as well as the opsonization of antigen were impaired in mutant mice. We conclude that complement activation by natural IgM cannot explain the role of C1q in primary antibody responses, but that endogenous, specific, wildtype IgM generated after immunization feedback-enhances the response to a booster dose of antigen. Importantly, this mechanism can only partially explain the role of complement in the generation of antibody responses because the IgG response was much lower in C3- or complement receptor 1 and 2-deficient mice than in C mu 13 mice.

Microglia, the immune cells of the central nervous system, are dynamic and heterogenous cells. While single cell RNA sequencing has become the conventional methodology for evaluating microglial state, transcriptomics do not provide insight into functional changes, identifying a critical gap in the field. Here, we propose a novel organelle phenotyping approach in which we treat live human induced pluripotent stem cell-derived microglia (iMGL) with organelle dyes staining mitochondria, lipids, lysosomes and acquire data by live-cell spectral microscopy. Dimensionality reduction techniques and unbiased cluster identification allow for recognition of microglial subpopulations with single-cell resolution based on organelle function. We validated this methodology using lipopolysaccharide and IL-10 treatment to polarize iMGL to an "inflammatory" and "anti-inflammatory" state, respectively, and then applied it to identify a novel regulator of iMGL function, complement protein C1q. While C1q is traditionally known as the initiator of the complement cascade, here we use organelle phenotyping to identify a role for C1q in regulating iMGL polarization via fatty acid storage and mitochondria membrane potential. Follow up evaluation of microglia using traditional read outs of activation state confirm that C1q drives an increase in microglia pro-inflammatory gene production and migration, while suppressing microglial proliferation. These data together validate the use of a novel organelle phenotyping approach and enable better mechanistic investigation of molecular regulators of microglial state.

IntroductionSynaptic loss is a hallmark of Alzheimer's disease (AD) that correlates with cognitive decline in AD patients. Complement-mediated synaptic pruning has been associated with this excessive loss of synapses in AD. Here, we investigated the effect of C5aR1 inhibition on microglial and astroglial synaptic pruning in two mouse models of AD.MethodsA combination of super-resolution and confocal and tridimensional image reconstruction was used to assess the effect of genetic ablation or pharmacological inhibition of C5aR1 on the Arctic48 and Tg2576 models of AD.ResultsGenetic ablation or pharmacological inhibition of C5aR1 partially rescues excessive pre-synaptic pruning and synaptic loss in an age and region-dependent fashion in two mouse models of AD, which correlates with improved long-term potentiation (LTP).DiscussionReduction of excessive synaptic pruning is an additional beneficial outcome of the suppression of C5a-C5aR1 signaling, further supporting its potential as an effective targeted therapy to treat AD.HighlightsC5aR1 ablation restores long-term potentiation in the Arctic model of AD. C5aR1 ablation rescues region specific excessive pre-synaptic loss. C5aR1 antagonist, PMX205, rescues VGlut1 loss in the Tg2576 model of AD. C1q tagging is not sufficient to induce VGlut1 microglial ingestion. Astrocytes contribute to excessive pre-synaptic loss at late stages of the disease.

The global malaria burden continues to remain high with most deaths occurring in young children. Understanding the protective immune mechanisms against malaria is imperative for the development of a highly efficacious vaccine since RTS,S and R21 which are recommended malaria vaccines, have modest efficacy and are limited to young children. Recent reports indicate that neutrophils may potentially play a key role in protective immunity against malaria via antibody-mediated interactions with Fc-gamma receptors (FcgR) expressed on their surface. Additionally, complement proteins may promote parasite clearance via interaction with antibodies. This thesis presents new findings on antibody interactions with complement (C1q) and FcgRs on neutrophils in immunity against severe malaria. It further presents new information on neutrophil phenotypes in response to naturally acquired malaria as well as phenotypes and transcription profiles in controlled human malaria infections (CHMI), and in vitro models. First, cytophilic IgG1 and IgG3 antibody subclasses, and antibodies that can fix C1q, or engage FcgRs to induce phagocytic clearance of merozoites were investigated. Additionally, opsonic phagocytosis of merozoites by neutrophils was quantified. These investigations were carried out on samples from young Papua New Guinean (PNG) children (n = 383) presenting with severe malaria versus uncomplicated malaria. In children presenting with severe malaria, cytophilic antibodies, antibodies that can fix C1q, or engage FcgRs against whole merozoites and neutrophil phagocytosis of merozoites opsonized by serum antibodies were lower than children with uncomplicated malaria. There was a strong association between high antibody responses to merozoites that fix complement or bind FcgRs, and reduced odds of severe disease. Second, cryopreserved neutrophils from children and adults in PNG, were phenotyped by examining receptors relevant to functions and malaria immunity. Neutrophils from child

The contribution of the gut microbiome to neuroinflammation, cognition, and Alzheimers disease progression has been highlighted over the past few years. Additionally, inhibition of various components of the complement system has repeatedly been demonstrated to reduce neuroinflammation and improve cognitive performance in AD mouse models. Whether the deletion of these complement components is associated with distinct microbiome composition, which could impact neuroinflammation and cognitive performance in mouse models has not yet been examined. Here, we provide a comprehensive analysis of conditional and constitutive knockouts, pharmacological inhibitors, and various housing paradigms for the animal models and wild-type controls at various ages. We aimed to determine the impact of C1q or C5aR1 inhibition on the microbiome in the Arctic and Tg2576 mouse models of AD, which develop amyloid plaques at different ages and locations. Analysis of fecal samples from WT and Arctic mice following global deletion of C1q demonstrated significant alterations to the microbiomes of Arctic but not WT mice, with substantial differences in abundances of Erysipelotrichales, Clostridiales and Alistipes. While no differences in microbiome diversity were detected between cohoused wildtype and Arctic mice with or without the constitutive deletion of the downstream complement receptor, C5aR1, a difference was detected between the C5aR1 sufficient (WT and Arctic) and deficient (C5ar1KO and ArcticC5aR1KO) mice, when the mice were housed segregated by C5aR1 genotype. However, cohousing of C5aR1 sufficient and deficient wildtype and Arctic mice resulted in a convergence of the microbiomes and equalized abundances of each identified order and genus across all genotypes. Similarly, pharmacologic treatment with the C5aR1 antagonist, PMX205, beginning at the onset of beta-amyloid plaque deposition in the Arctic and Tg2576 mice, demonstrated no impact of C5aR1 inhibition on the microbiome. This stud

The myelin sheath in the central nervous system (CNS) provides insulation and nutritional support for axons, facilitating rapid and efficient impulse conduction. Dysregulation of CNS myelination can lead to severe neurological disorders, such as leukodystrophies and multiple sclerosis. Microglia, the resident macrophages in the CNS, play crucial roles in myelin development, though the exact mechanisms are not fully understood. This study was conducted to understand the specific involvement of microglia in CNS myelination. Initial experiments involving the culture of neural stem cells showed that myelin sheath formation only occurred when a microglia-conditioned medium was present. This indicates a critical role of microglia secretomes in in vitro myelination. Moreover, the absence of C1q from the microglia secretomes resulted in myelination failure in the neural stem cell culture. This could be rectified by adding the native human C1q protein, suggesting that C1q is both required and sufficient for in vitro myelination. Further in vivo experiments were carried out to better understand the involvement of C1q in developmental myelination. By mapping C1q expression profiles, the results showed that the spatiotemporal pattern of C1q expression was similar to that of myelin proteins during early brain development. The use of a C1q conditional knockout mouse model (C1qaFL/FL: Cx3cr1CreER) revealed that C1q deficiency prior to the onset of myelination led to a significant reduction in myelin thickness and an increase in the g-ratio. Additionally, C1q deficiency resulted in a less precise alignment between myelin and axons, while the total myelin production remained unchanged. This suggests that C1q may be involved in the association between myelin and axons. In summary, these findings underscore the crucial role of microglia-derived C1q in developmental myelination. This discovery could potentially pave the way for new therapeutic strategies for treating demyelinating dise

Triggering receptor expressed on myeloid cells 2 (TREM2) is strongly linked to Alzheimer's disease (AD) risk, but its functions are not fully understood. Here, we found that TREM2 specifically attenuated the activation of classical complement cascade via high-affinity binding to its initiator C1q. In the human AD brains, the formation of TREM2-C1q complexes was detected, and the increased density of the complexes was associated with lower deposition of C3 but higher amounts of synaptic proteins. In mice expressing mutant human tau, Trem2 haploinsufficiency increased complement-mediated microglial engulfment of synapses and accelerated synaptic loss. Administration of a 41-amino-acid TREM2 peptide, which we identified to be responsible for TREM2 binding to C1q, rescued synaptic impairments in AD mouse models. We thus demonstrate a critical role for microglial TREM2 in restricting complement-mediated synaptic elimination during neurodegeneration, providing mechanistic insights into the protective roles of TREM2 against AD pathogenesis. © 2023 Elsevier Inc.

Antibody-dependent enhancement (ADE) of infection is generally known for many viruses. A potential risk of ADE in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has also been discussed since the beginning of the coronavirus disease 2019 (COVID-19) pandemic; however, clinical evidence of the presence of antibodies with ADE potential is limited. Here, we show that ADE antibodies are produced by SARS-CoV-2 infection and the ADE process can be mediated by at least two different host factors, Fcg receptor (Fcg R) and complement component C1q. Of 89 serum samples collected from acute or convalescent COVID-19 patients, 62.9% were found to be positive for SARS-CoV-2-specific IgG. Fcg R- and/or C1q-mediated ADE were detected in 50% of the IgG-positive sera, whereas most of them showed neutralizing activity in the absence of Fcg R and C1q. Importantly, ADE antibodies were found in 41.4% of the acute COVID-19 patients. Neutralizing activity was also detected in most of the IgG-positive sera, but it was counteracted by ADE in subneutralizing conditions in the presence of Fcg R or C1q. Although the clinical importance of ADE needs to be further investigated with larger numbers of COVID-19 patient samples, our data suggest that SARS-CoV-2 utilizes multiple mechanisms of ADE. C1q-mediated ADE may particularly have a clinical impact since C1q is present at high concentrations in plasma and its receptors are ubiquitously expressed on the surfaces of many types of cells, including respiratory epithelial cells, which SARS-CoV-2 primarily infects. IMPORTANCE Potential risks of antibody-dependent enhancement (ADE) in the coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has been discussed and the proposed mechanism mostly depends on the Fc gamma receptor (Fcg R). However, since Fcg Rs are exclusively expressed on immune cells, which are not primary targets of SARS-CoV-2, the clinical importance of

Pre-eclampsia is a pregnancy complication characterized by defective vascular remodeling in maternal decidua responsible for reduced blood flow leading to functional and structural alterations in the placenta. We have investigated the contribution of the complement system to decidual vascular changes and showed that trophoblasts surrounding unremodeled vessels prevalent in preeclamptic decidua fail to express C1q that are clearly detected in cells around remodeled vessels predominant in control placenta. The critical role of C1q is supported by the finding that decidual trophoblasts of female C1qa-/- pregnant mice mated to C1qa+/+ male mice surrounding remodeled vessels express C1q of paternal origin. Unlike C1qa-/- pregnant mice, heterozygous C1qa+/- and wild type pregnant mice share a high percentage of remodeled vessels. C1q was also found in decidual vessels and stroma of normal placentae and the staining was stronger in preeclamptic placentae. Failure to detect placental deposition of C1r and C1s associated with C1q rules out complement activation through the classical pathway. Conversely, the intense staining of decidual endothelial cells and villous trophoblast for ficolin-3, MASP-1 and MASP-2 supports the activation of the lectin pathway that proceeds with the cleavage of C4 and C3 and the assembly of the terminal complex. These data extend to humans our previous findings of complement activation through the lectin pathway in an animal model of pre-eclampsia and provide evidence for an important contribution of C1q in decidual vascular remodeling.

The contribution of the complement system to non-specific host defence and maintenance of homeostasis is well appreciated. Many particulate systems trigger complement activation but the underlying mechanisms are still poorly understood. Activation of the complement cascade could lead to particle opsonisation by the cleavage products of the third complement protein and might promote inflammatory reactions. Antibody binding in a controlled manner and/or sensing of particles by the complement pattern-recognition molecules such as C1q and mannose-binding lectin can trigger complement activation. Particle curvature and spacing arrangement/periodicity of surface functional groups/ligands are two important parameters that modulate complement responses through multivalent engagement with and conformational regulation of surface-bound antibodies and complement pattern-recognition molecules. Thus, a better fundamental understanding of nanometer- and angstrom-scale parameters that modulate particle interaction with antibodies and complement proteins could portend new possibilities for engineering of particulate drug carriers and biomedical platforms with tuneable complement responses and is discussed here.

My doctoral research focuses on uncovering how an overactive immune system arrests neurogenesis during aging and leads to cognitive problems. Neurogenesis is the process by which new neurons are born, migrate, and integrate into existing networks, and is essential for the maintenance of cognitive flexibility. As neurogenesis declines with age, brain networks become more vulnerable to inflammation. One large component of this inflammatory response in the aged brain is complement protein C1q, which is deposited within the brain during aging and can directly alter new neuron properties, such as migration, differentiation, metabolism, and cellular quiescence. Through in vitro studies, the first chapter of my dissertation describes how brain-relevant concentrations of C1q can modulate endogenous NSC behavior. The second chapter investigates how C1q inhibits neurogenesis and exploration of aged mice through a global C1q KO model. The third component of my dissertation asks whether the source of C1q, either from blood or locally produced in the brain by microglia, is important to its effects. Lastly, through a transplantation paradigm, the fourth component of my thesis investigates a receptor-mediated mechanism by which C1q alters the behavior of new neurons and therefore potential network integration or support for cognition. We discovered that C1q alters NSC characteristics in aged mice, contributes to aging-related behavior deficits, and can be rescued with via C1q KO. These studies contribute to the neuroimmunology field by identifying novel mechanisms that the immune system uses to modulate neurogenesis, thereby opening the opportunity for specific therapies to target cognitive decline and prevent age-related neurodegenerative disease.

The complement system, an essential part of the innate immune system, is composed of a group of secreted and membrane proteins that collectively participate in maintaining the function of the healthy and diseased brain. However, an inappropriate activation of the complement system has been related to an inflammatory response in multiple diseases, such as stroke, traumatic brain injury, multiple sclerosis, and Alzheimer's disease, as well as Zika infection and radiotherapy. In addition, C1q and C3 (initial activation components of the complement cascade) have been shown to play a key beneficial role in the refinement of synaptic circuits during developmental stages and adult plasticity. Nevertheless, excessive synaptic pruning in the adult brain can be detrimental and has been associated with synaptic loss in several pathological conditions. In this brief review, we will discuss the role of the complement system in synaptic pruning as well as its contribution to neurodegeneration and cognitive deficits. We also mention potential therapeutic approaches to target the complement system to treat several neuroinflammatory diseases and unintended consequences of radiotherapy.

Monoclonal antibodies directed against the CD20 surface antigen on B cells are widely used in the therapy of B cell malignancies. Upon administration, the antibodies bind to CD20 expressing B cells and induce their depletion via cell- and complement-dependent cytotoxicity or by induction of direct cell killing. The three antibodies currently most often used in the clinic are Rituximab (RTX), Ofatumumab (OFA) and Obinutuzumab (OBI). Even though these antibodies are all of the human IgG1 subclass, they have previously been described to vary considerably in the effector functions involved in therapeutic B cell depletion, especially in regards to complement activation. Whereas OFA is known to strongly induce complement-dependent cytotoxicity, OBI is described to be far less efficient. In contrast, the role of complement in RTX-induced B cell depletion is still under debate. Some of this dissent might come from the use of different in vitro systems for characterization of antibody effector functions. We therefore set out to systematically compare antibody as well as C1q binding and complement-activation by RTX, OFA and OBI on human B cell lines that differ in expression levels of CD20 and complement-regulatory proteins as well as human primary B cells. Applying real-time interaction analysis, we show that the overall strength of C1q binding to live target cells coated with antibodies positively correlated with the degree of bivalent binding for the antibodies to CD20. Kinetic analysis revealed that C1q exhibits two binding modes with distinct affinities and binding stabilities, with exact numbers varying both between antibodies and cell lines. Furthermore, complement-dependent cell killing by RTX and OBI was highly cell-line dependent, whereas the superior complement-dependent cytotoxicity by OFA was independent of the target B cells. All three antibodies were able to initiate deposition of C3b on the B cell surface, although to varying extent. This suggests that complem

Accurate determination of complement component C1q is hampered by the fact that C1q is an immune complex binding protein. Consequently, immunochemical techniques which rely on immune complex formation in fluid phase such as nephelometry and turbidimetry tend to give results which differ from those obtained by, for example, ELISA and other solid phase-based assays. In this chapter, we discuss the pros and cons of different techniques for the quantification of C1q and present a comprehensive protocol for a newly developed magnetic bead-based sandwich immunoassay which has replaced nephelometry in our complement diagnostic laboratory at the University Hospital in Uppsala., This work was supported by grant 2016-2075-5.1 and 2016-04519 from the Swedish Research Council (VR), the Medical Research Council of Southeast Sweden (FORSS), and by faculty grants from the Linnaeus university. The study was also supported by grants for development and validation of complement assays provided by the University Hospital in Uppsala, Sweden.

Movement is an essential behavior that is controlled by motor circuits. Within spinal motor circuits, motor neurons bridge the central and peripheral nervous systems by conveying central commands to the skeletal muscles. The precise assembly and refinement of spinal motor circuits is required for normal motor behavior. It is known for several brain regions that during development a larger than needed number of synapses are produced and subsequently eliminated. However, the mechanisms responsible for spinal motor circuit refinement and synapse maintenance are poorly understood. Similarly, the molecular mechanisms by which gene mutations cause dysfunction and elimination of synapses in neurodegenerative diseases that occur during the development are unknown. Complement proteins have emerged as critical mediators of synaptic refinement and circuit plasticity. Therefore, this study aimed to address whether complement proteins are involved in the synaptic refinement of spinal sensory-motor circuitry. Additionally, to address whether similar mechanisms are responsible for elimination of synapses in neurodegeneration, spinal muscular atrophy (SMA) was utilized as a model disease. SMA is a neurodegenerative disease that occurs during early development. It is caused by reduced levels of the ubiquitously expressed SMN protein. The hallmarks of SMA are loss of motor neurons, muscle atrophy and abnormal postural reflexes. It has been previously shown that decreased number of select synapses and sensory-motor circuit dysfunction precede motor neuron loss. The mechanisms leading to this selective synapse loss on SMN deficient motor neurons remained unknown. This study investigated whether complement-dependent mechanisms are involved in synapse elimination in SMA. Results of this study demonstrated that the complement protein C1q – initiating protein of classical pathway, is required for the refinement of spinal sensory-motor circuits during the normal development. Synaptic densit, Кретање је есенцијално понашање које је контролисано од стране моторних неуронских мрежа. У оквиру кичмених моторних мрежа, моторни нурони служе као мост између централног и периферног нервног система преносећи централне команде скелетним мишићима. Прецизно успостављање и преуређивање кичмених моторних мрежа је неопходно за нормално моторичко понашање. Познато је да се током развића појединих деловима мозга формира више него што је потребно синапси које касније бивају елиминисане. Међутим, механизми који су одговорни за преуређивање кичмених моторних мрежа и за одржавање синапси недовољно су испитани. Слично, молекуларни механизми одговорни за генским мутацијама изазвану дисфункцију и елиминацију синапси у неуродегенеративним болестима које се јављају током развића тренутно су непознати. Протеини система комплемента су се показали као критични медијатори финог синаптичког подешавања и пластичности неуронских мрежа. Стога је ова студија имала за циљ да испита да ли су протеини комплемент система умешани у синаптичкo преуређивање кичмене сензорно-моторне неуронске мреже. Додатно, не би ли се испитало да ли су слични механизми одговорни за елиминацију синапси током неуродегенерације, спинална мишићна атрофија (СМА) је коришћена као модел неуродегенартивнe болести. СМА је неуродегенеративна болест која се јавља рано током развића. Изазивана је смањењем нивоа убиквитно експримираног SMN (енг. survival motor neuron) протеина. Карактеристике СМА су губитак моторних неурона, атрофија мишића и абнормални постурални рефлекси. Претходно је показано да снижени бројеви одређених синапси и дисфункција сензорно-моторне неуронске мреже предходе губитку моторних неурона. Механизми који доводе до овог селективног губитка синапси на моторним неуронима којима недостаје SMN тренутно су непознати. У овој студији је испитано да ли су комплемент-зависни мезанизми умешани у елиминацију синапси у СМА. класичног пута, неопходан за преуређивање кичмених сензорно-моторних мрежа током нормалног

C1q is an important recognition protein in the complement system, which is a major protein cascade in the innate immune system. Upon recognition of a target by C1q, the target is marked for opsonization and destruction. C1q recognizes many pathogenic patterns directly, but an important target is the Fc domain of antibodies binding to their antigen. In this paper, the curvature-dependence of the interaction between IgG and C1q is studied by surface plasmon resonance and quartz crystal microbalance. IgG is organized in similar surface coverage densities on flat polystyrene surfaces and polystyrene nanoparticles of different sizes, and the amount of C1q binding to the IgG is investigated. Nanoparticles in solution were found to aggregate upon incubation with IgG, and therefore a new technique utilizing nanoparticles binding to antifouling polymer brush functionalized surfaces was used to prepare surfaces with nanoparticles for measurements with surface plasmon resonance. Interestingly antigen-bound IgG at the curved surface of nanoparticles showed 5.6 times lower binding of C1q compared to at matched flat surfaces. There was no significant difference between the binding at 100 and 200 nm polystyrene particles. These findings are important for designing drug delivery systems to evade the complement system.

To assess if markers of complement activation are associated with disease activity, C1q, C3, C3a and sC5b-9 levels in plasma and cerebrospinal fluid (CSF) were determined in 41 patients with clinically isolated syndrome (CIS) or remitting multiple sclerosis (RRMS), in a prospective longitudinal four-year cohort study. C1q in CSF (CSF-C1q) was significantly higher in patients than in controls. Baseline CSF-C1q and CSF-C3a correlated with several neuroinflammatory markers and neurofilament light chain levels. Baseline CSF-C3a correlated with the number of T2 lesions at baseline and new T2 lesions during follow-up. Baseline CSF-C3a was also significantly higher in patients with (n = 21) than in patients without (n = 20) signs of disease activity according to the NEDA-3 concept during one year of follow-up (p <= .01) Study results support that complement activation is involved in MS pathophysiology and that CSF-C3a carries prognostic information.

Objectives: C1q is a valuable biomarker of disease activity in systemic lupus erythematosus (SLE). The "gold standard" assay, rocket immunoelectrophoresis (RIE), is time-consuming, and thus a shift to soluble immune precipitation techniques such as nephelometry has occurred. However, quantification of C1q with these techniques has been questioned as a result of the antibody binding properties of C1q. In the present work, we have compared results using various techniques (RIE, nephelometry, and ELISA) and have developed and validated a new magnetic bead-based sandwich immunoassay (MBSI). Methods: C1q was quantified by nephelometry and the new sandwich immunoassay in 45 serum samples analyzed using RIE. C1q was also assessed in plasma using RIE and sandwich immunoassay in samples from SLE patients with nephritis (n = 69), SLE patients without nephritis (n = 310) as classified by BILAG score, and matched controls (n = 322). In addition, cerebrospinal fluid (CSF) samples from 31 patients, previously analyzed with ELISA, were also analyzed with the MBSI to test the behavior of this new assay in the lower detection range. Results: We found a strong correlation between the new MBSI, RIE, and ELISA, but not with nephelometry. The MBSI demonstrated lower levels of C1q in SLE patients than in matched controls (p < 0.0001), and patients with nephritis had lower levels than patients without nephritis (p < 0.01). Similarily, RIE showed significant differences between the patient groups (p < 0.0001). An association was also found between the levels of C1q and the SLE disease activity index (SLEDAI). Furthermore, there was good correlation between the values obtained by MBSI and ELISA, in both serum (r = 0.960) and CSF (r = 0.786), underscoring the ability of both techniques to measure low concentrations of C1q with high accuracy. Conclusion: The sandwich immunoassay correlated well with RIE, but soluble immune precipitation techniques, such as nephelometry, did not appea

Laboratory analyses of human body fluids play an important role in clinical diagnosis. This thesis comprises projects in which various immune assays have been developed and evaluated as complement diagnostics in both plasma and cerebrospinal fluid (CSF). Various methods have been used, such as ELISA, Western blot, flow cytometry, and xMAP technology. In paper 1, we monitored complement parameters in EDTA-plasma and CSF from patients with suspected neuroborreliosis (NB) by using in-house sandwich ELISAs. We found significantly elevated levels of C1q, C4, C3, and C3a in CSF, but not in plasma, suggesting that complement plays a role in the intrathecal immune response in NB. Complement is a main player in early inflammation, and in paper 2, we investigated the role of complement activation in phagocytosis and the release of cytokines and chemokines in response to two clinical isolates: Borrelia afzelii K78 and Borrelia garinii LU59. Our results show that complement activation plays an important role in the initial process of phagocytosis, but not in the subsequent cytokine release that occurs in response to live Borrelia spirochetes. C1q, a valuable biomarker of disease activity in systemic lupus erythematosus (SLE), can be quantitated using a number of different immunochemical techniques. In paper 3, we developed and validated a magnetic bead-based immunoassay for quantifying C1q in EDTA-plasma and CSF. In contrast to soluble immunoprecipitation assays such as nephelometry and turbidimetry, this new assay was not hampered by the interaction between C1q and detecting antibodies. The novel assay was shown to give a clear correlation between nephritis and SLEDAI score in SLE. Warfarin is a commonly used but complicated treatment in patients with thrombosis. It reduces the function of vitamin K-dependent coagulation proteins, including protein S, which is a ligand for C4b-binding protein (C4BP). In paper 4, we demonstrated a decrease in various isoforms of C4BP that resu

In 2014, specific recommendations for complement nomenclature were presented by the complement field. There remained some unresolved designations and new areas of ambiguity, and here we propose solutions to resolve these remaining issues. To enable rapid understanding of the intricate complement system and facilitate therapeutic development and application, a uniform nomenclature for cleavage fragments, pattern recognition molecules (PRMs) and enzymes of the lectin pathway and regulatory proteins of the complement system are proposed, and a standardization of language to designate different activation states of complement components is recommended.

A unique strategy for effective, versatile, and facile surface biofunctionalization employing a recombinant spider silk protein genetically functionalized with the antibody-binding Z domain (Z-4RepCT) is reported. It is demonstrated that Z-silk can be applied to a variety of materials and platform designs as a truly one-step and chemical-free surface modification that site specifically captures antibodies while simultaneously reducing nonspecific adsorption. As a model surface, SiO2 is used to optimize and characterize Z-silk performance compared to the Z domain immobilized by a standard silanization method. First, Z-silk adsorption is investigated and verified its biofunctionality in a long-term stability experiment. To assess the binding capacity and protein-protein interaction stability of Z-silk, the coating is used to capture human antibodies in various assay formats. An eightfold higher binding capacity and 40-fold lower detection limit are obtained in the immunofluorescence assay, and the complex stability of captured antibodies is shown to be improved by a factor of 20. Applicability of Z-silk to functionalize microfluidic devices is demonstrated by antibody detection in an electrokinetic microcapillary biosensor. To test Z-silk for biomarker applications, real-time detection and quantification of human immunoglobulin G are performed in a plasma sample and C1q capture from human serum using an anti-C1q antibody.

Traumatic brain injury (TBI) is of particular concern for the aging community since there is both increased incidence of TBI and decreased functional recovery in this population. In addition, TBI is the strongest environmental risk factor for development of Alzheimer's disease and other dementia-related neurodegenerative disorders. Critical changes that affect cognition take place over time following the initial insult. Our previous work identified immune system activation as a key contributor to cognitive deficits observed in aged animals. Using a focal contusion model in the current study, we demonstrate a brain lesion and cavitation formation, as well as prolonged blood⁻brain barrier breakdown. These changes were associated with a prolonged inflammatory response, characterized by increased microglial cell number and phagocytic activity 30 days post injury, corresponding to significant memory deficits. We next aimed to identify the injury-induced cellular and molecular changes that lead to chronic cognitive deficits in aged animals, and measured increases in complement initiation components C1q, C3, and CR3, which are known to regulate microglial⁻synapse interactions. Specifically, we found significant accumulation of C1q on synapses within the hippocampus, which was paralleled by synapse loss 30 days post injury. We used genetic and pharmacological approaches to determine the mechanistic role of complement initiation on cognitive loss in aging animals after TBI. Notably, both genetic and pharmacological blockade of the complement pathway prevented memory deficits in aged injured animals. Thus, therapeutically targeting early components of the complement cascade represents a significant avenue for possible clinical intervention following TBI in the aging population.

While the mechanisms underlying the functions of the complement system in the central nervous system (CNS) and systemically, namely opsonization, chemotaxis, membrane lysis, and regulation of inflammation are the same, the plethora of functions that complement orchestrates in the central nervous system (CNS) is complex. Strictly controlled expression of complement effector molecules, regulators and receptors across the gamut of life stages (embryogenesis, development and maturation, aging and disease) dictate fascinating contributions for this ancient system. Furthermore, it is becoming apparent that complement functions differ widely across distinct brain regions. This review provides a comprehensive overview of the newly identified roles for complement in the brain, including its roles in CNS development and function, during aging and in the processes of neurodegeneration. The diversity and selectively of beneficial and detrimental activities of complement, while challenging, should lead to precision targeting of specific components to provide disease modifying treatments for devastating psychiatric and neurodegenerative disorders that are still without effective treatment.

An intact complement system is essential for the initiation of a normal antibody response. Antibodies can regulate their own production against the antigens that they are specific for. Both IgG3 and IgM are able to enhance the antibody response via complement. Here, we have compared the fate of OVA-TNP (ovalbumin-2,4,6-trinitrophenyl) administered intravenously to mice either alone or in complex with monoclonal IgG3 anti-TNP. IgG3-antigen complexes bind to marginal zone (MZ) B cells via complement receptors 1 and 2 (CR1/2) and are transported into splenic follicles. The majority (50% - 90%) of the antigens is deposited on follicular dendritic cells (FDC) and the antigen distribution pattern is strikingly similar to peripheral dendrites/processes of FDC already 2 h after immunization. The development of germinal centers (GC) induced by IgG3-antigen complexes is impaired in mice lacking CR1/2. Experiments on bone marrow chimeric mice show that CR1/2 expression on both MZ B cells and FDC is required for optimal IgG3-mediated enhancement of antibody responses. Complement factors C3 and C1q are essential for OVA-TNP delivery and deposition on splenic FDC. The production of IgG anti-OVA is abrogated in mice lacking CR1/2, C1q, and C3. Further, IgG3-antigen complexes dramatically upregulate the memory response against OVA-TNP by inducing OVA-specific memory cells. Besides small protein OVA, IgG3 can also upregulate humoral responses against large soluble keyhole limpet hemocyanin. To further study the role of MZ B-cells and CR1/2 in enhancement of antibody responses, a knock-in mouse strain, Cμ13, was used. IgM in this mouse strain is unable to activate complement due to a point mutation in the constant µ-heavy chain. Cμ13 mice have a higher proportion of MZ B cells, with higher CR1/2 expression, than wild-type mice. More IgG3-immune complexes are captured by MZ B cells and deposited on FDC in Cμ13 than in WT mice. In spite of this, IgG3 did not enhance the primary antibod

BackgroundThe complement cascade not only provides protection from infection but can also mediate destructive inflammation. Complement is also involved in elimination of neuronal synapses which is essential for proper development, but can be detrimental during aging and disease. C1q, required for several of these complement-mediated activities, is present in the neuropil, microglia, and a subset of interneurons in the brain.MethodsTo identify the source(s) of C1q in the brain, the C1qa gene was selectively inactivated in the microglia or Thy-1+ neurons in both wild type mice and a mouse model of Alzheimer's disease (AD), and C1q synthesis assessed by immunohistochemistry, QPCR, and western blot analysis.ResultsWhile C1q expression in the brain was unaffected after inactivation of C1qa in Thy-1+ neurons, the brains of C1qa FL/FL :Cx3cr1 CreERT2 mice in which C1qa was ablated in microglia were devoid of C1q with the exception of limited C1q in subsets of interneurons. Surprisingly, this loss of C1q occurred even in the absence of tamoxifen by 1 month of age, demonstrating that Cre activity is tamoxifen-independent in microglia in Cx3cr1 CreERT2/WganJ mice. C1q expression in C1qa FL/FL : Cx3cr1 CreERT2/WganJ mice continued to decline and remained almost completely absent through aging and in AD model mice. No difference in C1q was detected in the liver or kidney from C1qa FL/FL : Cx3cr1 CreERT2/WganJ mice relative to controls, and C1qa FL/FL : Cx3cr1 CreERT2/WganJ mice had minimal, if any, reduction in plasma C1q.ConclusionsThus, microglia, but not neurons or peripheral sources, are the dominant source of C1q in the brain. While demonstrating that the Cx3cr1 CreERT2/WganJ deleter cannot be used for adult-induced deletion of genes in microglia, the model described here enables further investigation of physiological roles of C1q in the brain and identification of therapeutic targets for the selective control of complement-mediated activities contributing to neurodeg

Inflammation is a part of the initial process leading to atherosclerosis and cholesterol crystals (CC), found in atherosclerotic plaques, which are known to induce complement activation. The pentraxins C-reactive protein (CRP), long pentraxin 3 (PTX3), and serum amyloid P component (SAP) are serum proteins associated with increased risk of cardiovascular events and these proteins have been shown to interact with the complement system. Whether the pentraxins binds to CC and mediate downstream complement-dependent inflammatory processes remains unknown. Binding of CRP, PTX3, and SAP to CC was investigated in vitro by flow cytometry and fluorescence microscopy. CRP, PTX3, and SAP bound to CC in a concentration-dependent manner. CRP and PTX3 interacted with the complement pattern recognition molecule C1q on CC by increasing the binding of both purified C1q and C1q in plasma. However, CRP was the strongest mediator of C1q binding and also the pentraxin that most potently elevated C1q-mediated complement activation. In a phagocytic assay using whole blood, we confirmed that phagocytosis of CC is complement dependent and initiated by C1q-mediated activation. The pathophysiological relevance of the in vitro observations was examined in vivo in human atherosclerotic plaques. CRP, PTX3, and SAP were all found in atherosclerotic plaques and were located mainly in the cholesterol-rich necrotic core, but co-localization with the terminal C5b-9 complement complex was only found for CRP. In conclusion, this study identifies CRP as a strong C1q recruiter and complement facilitator on CC, which may be highly relevant for the development of atherosclerosis.

Cholera epidemics are caused by Vibrio cholerae serogroups O1 and O139, whereas strains collectively known as non-O1/non-O139 V. cholerae are found in cases of extraintestinal infections and bacteremia. The mechanisms and factors influencing the occurrence of bacteremia and survival of V. cholerae in normal human serum have remained unclear. We found that naturally occurring IgG recognizing V. cholerae outer membrane protein U (OmpU) mediates a serum-killing effect in a complement C1q-dependent manner. Moreover, outer membrane vesicles (OMVs) containing OmpU caused enhanced survival of highly serum-sensitive classical V. cholerae in a dose-dependent manner. OMVs from wild-type and ompU mutant V. cholerae thereby provided a novel means to verify by extracellular transcomplementation the involvement of OmpU. Our data conclusively indicate that loss, or reduced expression, of OmpU imparts resistance to V. cholerae towards serum killing. We propose that the difference in OmpU protein levels is a plausible reason for differences in serum resistance and the ability to cause bacteremia observed among V. cholerae biotypes. Our findings provide a new perspective on how naturally occurring antibodies, perhaps induced by members of the microbiome, may play a role in the recognition of pathogens and the provocation of innate immune defense against bacteremia.

Systemic lupus erythematosus is a chronic autoimmune disease with multifactorial ethiopathogenesis. The complement system is involved in both the early and late stages of disease development and organ damage. To better understand autoantibody mediated complement consumption we examined ex vivo immune complex formation on autoantigen arrays. We recruited patients with SLE (n = 211), with other systemic autoimmune diseases (n = 65) and non-autoimmune control subjects (n = 149). Standard clinical and laboratory data were collected and serum complement levels were determined. The genotype of SNP rs1143679 in the ITGAM gene was also determined. Ex vivo formation of immune complexes, with respect to IgM, IgG, complement C4 and C3 binding, was examined using a functional immunoassay on autoantigen microarray comprising nucleic acids, proteins and lipids. Complement consumption of nucleic acids increased upon binding of IgM and IgG even when serum complement levels were decreased due to consumption in SLE patients. A negative correlation between serum complement levels and ex vivo complement deposition on nucleic acid autoantigens is demonstrated. On the contrary, complement deposition on tested protein and lipid autoantigens showed positive correlation with C4 levels. Genetic analysis revealed that the non-synonymous variant rs1143679 in complement receptor type 3 is associated with an increased production of anti-dsDNA IgG antibodies. Notwithstanding, homozygous carriers of the previously reported susceptible allele (AA) had lower levels of dsDNA specific IgM among SLE patients. Both the non-synonymous variant rs1143679 and the high ratio of nucleic acid specific IgG/IgM were associated with multiple organ involvement. In summary, secondary complement deficiency in SLE does not impair opsonization of nucleic-acid-containing autoantigens but does affect other antigens and potentially other complement dependent processes. Dysfunction of the receptor recognizing complement o

Complement is present mainly in blood. However, following mechanical damage or inflammation, serous exudates enter the mucosal surfaces. Here, the complement proteins interact with other endogenous molecules to keep microbes from entering the parenteral tissues. One of the mucosal proteins known to interact with the early complement components of both the classical and the lectin pathway is the salivary scavenger and agglutinin (SALSA). SALSA is also known as deleted in malignant brain tumors 1 and gp340. It is found both attached to the epithelium and secreted into the surrounding fluids of most mucosal surfaces. SALSA has been shown to bind directly to C1q, mannose-binding lectin, and the ficolins. Through these interactions SALSA regulates activation of the complement system. In addition, SALSA interacts with surfactant proteins A and D, secretory IgA, and lactoferrin. Ulcerative colitis and Crohn's disease are examples of diseases, where complement activation in mucosal tissues may occur. This review describes the latest advances in our understanding of how the early complement components interact with the SALSA molecule. Furthermore, we discuss how these interactions may affect disease propagation on mucosal surfaces in immunological and inflammatory diseases.

Traumatic injury to CNS fiber tracts is accompanied by failure of severed axons to regenerate and results in lifelong functional deficits. The inflammatory response to CNS trauma is mediated by a diverse set of cells and proteins with varied, overlapping, and opposing effects on histological and behavioral recovery. Importantly, the contribution of individual inflammatory complement proteins to spinal cord injury (SCI) pathology is not well understood. Although the presence of complement components increases after SCI in association with axons and myelin, it is unknown whether complement proteins affect axon growth or regeneration. We report a novel role for complement C1q in neurite outgrowth in vitro and axon regrowth after SCI. In culture, C1q increased neurite length on myelin. Protein and molecular assays revealed that C1q interacts directly with myelin associated glycoprotein (MAG) in myelin, resulting in reduced activation of growth inhibitory signaling in neurons. In agreement with a C1q-outgrowth-enhancing mechanism in which C1q binding to MAG reduces MAG signaling to neurons, complement C1q blocked both the growth inhibitory and repulsive turning effects of MAG in vitro. Furthermore, C1q KO mice demonstrated increased sensory axon turning within the spinal cord lesion after SCI with peripheral conditioning injury, consistent with C1q-mediated neutralization of MAG. Finally, we present data that extend the role for C1q in axon growth and guidance to include the sprouting patterns of descending corticospinal tract axons into spinal gray matter after dorsal column transection SCI.

Inflammation plays a key role in the pathophysiology of Glioblastoma Multiforme (GBM). Here we focus on the contribution of the so far largely ignored complement system.ELISA and immunohistochemistry were combined to assess levels and localization of critical components of the initiation- and effector pathways of the complement cascade in sera and tumor tissue from GBM patients and matched controls.Serum levels of factor-B were decreased in GBM patients whereas C1q levels were increased. C1q and factor-B deposited in the tumor tissue. Deposition of C3 and C5b-9 suggests local complement activation. MBL deficiency, based on serum levels, was significantly less frequent among GBM patients compared to controls (14% vs. 33%). Therefore low levels of MBL may protect against the initiation/progression of GBM.

Conclusion: No significant differences in the number of immune aberrations were seen between children with or without severe recurrent acute otitis media (rAOM); however, subnormal values of immunological markers were found more often than expected, and 4 of the 60 children had treatment-requiring immune deficiencies. Objective: Minor immunologic aberrations have been reported to be more frequent in children with rAOM. Immune investigation is recommended in children with severe rAOM, defined as six or more AOM episodes per year. The purpose of this study was to describe immunological findings in young children at high risk of developing rAOM, and to relate these to the number of expected aberrations and to the presence of severe rAOM. Methods: A total of 109 children at risk of developing rAOM were offered immune investigation including complement function, immunoglobulins with subclasses and cellular immunity. Results: Sixty patients were tested, 31 of whom had severe rAOM and 12 of whom did not develop rAOM. Low levels of IgG2 (27%), C1q (31%) and mannan-binding lectin (21%) were found up to eight times as often as expected. Although subnormal values were more frequent among children with severe rAOM, the study was too small to provide reliable evidence of any difference. Four children were diagnosed with immune deficiencies that required treatment.

Deficiency in C1q, the recognition component of the classical complement cascade and a key opsonin involved in apoptotic cell clearance, leads to lupus-like autoimmune diseases characterized by auto-antibodies to self proteins and aberrant T and B cell activation. Studies have suggested that these pathological consequences may result from impaired clearance of apoptotic cells. To investigate how C1q may modulate inflammation resulting from diminished apoptotic cell clearance, I applied a novel system consisting entirely of primary human cells: human monocyte-derived macrophages (Mac) and dendritic cells (DC), ingesting autologous apoptotic human lymphocytes (C1q-polarized Mac; or C1q-polarized DC), and either autologous or allogeneic human T cells. This physiologically relevant experimental system enabled characterization of the C1q-polarized Mac or C1q-polarized DC functional phenotype and subsequent Mac and DC-mediated T cell activation, avoiding the caveats of other systems using either transformed cell lines or non-physiologic presentation of C1q. The results demonstrate that C1q influences intracellular signaling and gene expression of both secreted protein and cell surface receptors. That is, C1q-polarized Mac exhibited enhanced STAT1 phosphorylation that is correlated with attenuated NLRP3 inflammasome activation and sequential induction of type I IFN-s, IL-27, and IL-10 in LPS-stimulated Mac relative to Mac ingesting apoptotic lymphocytes alone. C1q-polarized DC also exhibited enhanced IL-27 expression relative to DC ingesting apoptotic lymphocytes without C1q. Under the same conditions C1q-polarized Mac showed suppressed induction of CD40 and enhanced expression of PD-L1 and PD-L2 expression while C1q-polarized DC exhibited reduced CD86 induction and elevated PD-L2 and CD39 expression relative to DC ingesting apoptotic cells alone. Finally, C1q-polarized Mac reduced allogeneic and autologous Th17 and Th1 subset proliferation, and initiated a trend towards i

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3776260, [EN] Background There is a medical need for diagnostic biomarkers in lung cancer. We evaluated the diagnostic performance of complement activation fragments. Methods We assessed complement activation in four bronchial epithelial and seven lung cancer cell lines. C4d, a degradation product of complement activation, was determined in 90 primary lung tumors; bronchoalveolar lavage supernatants from patients with lung cancer (n = 50) and nonmalignant respiratory diseases (n = 22); and plasma samples from advanced (n = 50) and early lung cancer patients (n = 84) subjects with inflammatory lung diseases (n = 133), and asymptomatic individuals enrolled in a lung cancer computed tomography screening program (n = 190). Two-sided P values were calculated by Mann-Whitney U test. Results Lung cancer cells activated the classical complement pathway mediated by C1q binding that was inhibited by phosphomonoesters. Survival was decreased in patients with high C4d deposition in tumors (hazard ratio [HR] = 3.06; 95% confidence interval [CI] = 1.18 to 7.91). C4d levels were increased in bronchoalveolar lavage fluid from lung cancer patients compared with patients with nonmalignant respiratory diseases (0.61 +/- 0.87 vs 0.16 +/- 0.11 mu g/mL; P < .001). C4d levels in plasma samples from lung cancer patients at both advanced and early stages were also increased compared with control subjects (4.13 +/- 2.02 vs 1.86 +/- 0.95 mu g/mL, P < 0.001; 3.18 +/- 3.20 vs 1.13 +/- 0.69 mu g/mL, P < .001, respectively). C4d plasma levels were associated with shorter survival in patients at advanced (HR = 1.59; 95% CI = 0.97 to 2.60) and early stages (HR = 5.57; 95% CI = 1.60 to 19.39). Plasma C4d levels were reduced after surgical removal of lung tumors (P < .001) and were associated with increased lung cancer risk in asymptomatic individuals with (n = 32) or without lung cancer (n = 158) (odds ratio = 4.38; 95% CI = 1.61 to 11.93). Conclusions Complement fragment C4d may serve as a biomarker for ear

Disturbances of host integrity have the potential to evoke activation of innate immunologic and hemostatic protection mechanisms in blood. Irrespective of whether the activating stimulus is typically immunogenic or thrombotic, it will generally affect both the complement system and platelets to a certain degree. The theme of this thesis is complement and platelet activity, which is intersected in all five included papers. The initial aim was to study the responses and mechanisms of the complement cascade in relation to platelet activation. The secondary aim was to use an applied approach to regulate platelets and complement on model biomaterial and cell surfaces. Complement activation was found in the fluid phase in response to platelet activation in whole blood. The mechanism was traced to platelet release of stored chondroitin sulfate-A (CS-A) and classical pathway activation via C1q. C3 was detected at the platelet surface, though its binding was independent of complement activation. The inhibitors factor H and C4-binding protein (C4BP) were detected on activated platelets, and their binding was partly dependent on surface-exposed CS-A. Collectively, these results showed that platelet activation induces inflammatory complement activation in the fluid phase. CS-A was shown to be a central molecule in the complement-modulatory functions of platelets by its interaction with C1q, C4BP, and factor H. Platelet activation and surface adherence were successfully attenuated by conjugating an ADP-degrading apyrase on a model biomaterial. Only minor complement regulation was seen, and was therefore targeted specifically on surfaces and cells by co-immobilizing a factor H-binding peptide together with the apyrase. This combined approach led to a synchronized inhibition of both platelet and complement activation at the interface of biomaterials/xenogeneic cells and blood. In conclusion, here presents a novel crosstalk-mechanism for activation of complement when triggering

In order to find a clue to the molecular evolution mechanism of collagen-like molecules, comparative studies on the function and the structure of human and bovine C1q, a collagen-like complement component, were carried out. The hemolytic activity of bovine C1q was conspicuously higher than that of human C1q. On the other hand, the ability of bovine C1q to bind to Fc of immune complexes was a little lower than that of human C1q. However, these activities were interchangeable between them. Both the N-terminal collagen-like (CLF) and the C-terminal globular fragments (GF) of these C1q were highly purified by enzymic digestion followed by gel filtration, and their physicochemical and antigenic characterization was performed. Polyacrylamide gel electrophoresis (PAGE) analyses have shown that not only whole molecules of both C1q but also CLFs and GFs seem to be composed of essentially the same peptide structures. Some similarities between amino acid compositions of both CLFs and great similarities between those of both GFs were found. Moreover, great similarities of amino acid compositions were found among three non-covalently linked chains of each GF as well as between the corresponding chains of both GFs. Immunodiffusion analyses and radioimmune inhibition tests have shown that the definitive antigenic cross-reactivity is present between these two C1q molecules, and that the regions participating in interspecies cross-reactions are located in both CLF and GF of C1q. These results suggest that both CLF and GF on the C1q molecule remained highly conserved in their evolution, and that the hemolytic activity and the Fc-binding ability evolved independently to some extent. The possible relation of the C1q evolution to that of collagen molecules has been also discussed.

Blood platelets express several receptors involved in immunity (e.g. complement-, toll-like- and Fc gamma-receptors) and release inflammatory mediators. Furthermore, formation of platelet-leukocyte aggregates has an important role during inflammatory conditions such as coronary artery disease. Thus, apart from their well-known role in haemostasis, platelets are today also recognized as cells with immunomodulatory properties. We have previously reported regulatory effects of complement protein 1q (C1q) on platelet activation in experimental setups using isolated cells. In the present study we have proceeded by investigating effects of C1q on collagen-induced aggregation, production of reactive oxygen species (ROS), formation of platelet-leukocyte aggregates and levels of soluble P-selectin in whole blood. Impedance measurements showed that C1q inhibited collagen-induced aggregation whereas it potentiated the collagen-provoked production of ROS in a luminol-dependent chemiluminescence assay. The effects of C1q on aggregation and ROS-production were dependent upon platelets, as they were no longer observed in presence of the platelet (GpIIb/IIIa) inhibitor Reopro. Furthermore, the levels of soluble P-selectin were found to be lowered upon treatment with C1q prior to addition of collagen. There was also a trend towards a decreased formation of large platelet-leukocyte aggregates in collagen-stimulated whole blood following C1q treatment. In conclusion, our data indicate that C1q could have a role in regulating platelet activation and associated leukocyte recruitment during vessel wall injury. This has implications for inflammatory disorders such as coronary artery disease., Funding Agencies:strategic research area "Materials in Medicine" County Council of Östergötland Swedish Research Council Swedish Society of Medicine Swedish Fund for Research Without Animal Experiments Eleanora Demoroutis Nanna Svartz foundation Magn. Bergvall foundation Lars Hiertas foundation Goljes research foundation

Background: Complement proteins and activation products have been found associated with neuropathology in Alzheimer's disease (AD). Recently, a C5a receptor antagonist was shown to suppress neuropathology in two murine models of AD, Tg2576 and 3xTg. Previously, a genetic deficiency of C1q in the Tg2576 mouse model showed an accumulation of fibrillar plaques similar to the complement sufficient Tg2576, but reactive glia were significantly decreased and neuronal integrity was improved suggesting detrimental consequences for complement activation in AD. The goal of this study was to define the role of the classical complement activation pathway in the progression of pathology in the 3xTg mouse that develops tangles in addition to fibrillar plaques (more closely reflecting human AD pathology) and to assess the influence of complement in a model of AD with a higher level of complement hemolytic activity. Methods: 3xTg mice deficient in C1q (3xTgQ-/-) were generated, and both 3xTg and 3xTgQ-/- were backcrossed to the BUB mouse strain which has higher in vitro hemolytic complement activity. Mice were aged and perfused, and brain sections stained for pathological markers or analyzed for proinflammatory marker expression. Results: 3xTgQ-/- mice showed similar amounts of fibrillar amyloid, reactive glia and hyperphosphorylated tau as the C1q-sufficient 3xTg at the ages analyzed. However, 3xTg and 3xTgQ-/- on the BUB background developed pathology earlier than on the original 3xTg background, although the presence of C1q had no effect on neuropathological and pro-inflammatory markers. In contrast to that seen in other transgenic models of AD, C1q, C4 and C3 immunoreactivity was undetectable on the plaques of 3xTg in any background, although C3 was associated with reactive astrocytes surrounding the plaques. Importantly, properdin a component of the alternative complement pathway was associated with plaques in all models. Conclusions: In contrast to previously investigated tran

In this thesis the evolution of the sghC1q gene family is explored throughout the metazoan lineage and within the zebrafish (Danio rerio) genome. This involved novel bioinformatic analyses, extensive synthesis of the literature, development of a bioinformatic tool, and the transcriptional assessment of the full complement of sghC1q genes within \emph{D. rerio} during infection and early development.The secreted globular head C1q (sghC1q) genes can be characterized as a family of genetic loci each encoding a signal peptide followed by a complement component 1q globular (gC1q) motif. Members of this family have been referred to as precerebellin-like (cblnl), C1q-like or ovary specific C1q-like factors. Previous studies have found gene family members in multiple organisms with varying numbers of copies within a species. The genes are known to be transcribed in response to infection and/or during development.The domain of the C1q globular head (gC1q or ghC1q) appears to be ancient; present even in prokaryotes. With increasing complexity of organisms, this domain can sometimes be found accompanying first a signal peptide motif (indicative of secretion), and later with a collagen region. A comprehensive naming scheme is suggested based on these evolutionary adaptations. Computational modeling shows the globular head to be structurally conserved throughout the metazoa.The EST Keeper program was developed to facilitate these studies in identification of sets of non-redundant homologous genes from BLAST results that often contain redundant copies and gene fragments. It was built as a Flash based webservice and can be used to find gene families within genomes and EST datasets.Twenty sghC1q genes were found in the D. rerio genome (Zv9) and transcriptionally assessed. Two of the examined twenty genes showed significant up-regulation within 24 h of infection with the fish pathogen Streptococcus iniae, and eleven were expressed during early development. Due to the clustered natur

Several studies have shown that complement and thrombotic events co-exist. Platelets have been suspected to act as the bridge between the two cascade systems. To study the platelet-induced complement activation we developed a system in which platelets were activated by thrombin receptor activating peptide (TRAP) in platelet rich plasma (PRP) or whole blood anti-coagulated using the specific thrombin inhibitor, lepirudin. TRAP-activated platelets induced a fluid-phase complement activation measured as generation of C3a and sC5b-9, triggered by released chondroitin sulphate-A (CS-A) which interacted with C1q and activated the complement system through the classical pathway. Complement components C1q, C3, C4 and C9 were also shown to bind to TRAP-activated platelets but this binding did not seem to be due to a complement activation since blocking of complement activation at the C1q or C3 levels did not affect the binding of the complement proteins. The C3 which bound to activated platelets consisted of C3(H2O), indicating that bound C3 was not proteolytically activated. Binding of C1q was partially dependent on CS-A exposure on activated platelets. The abolished complement activation on the surface of activated platelets was suggested to be dependent on the involvement of several complement inhibitors. We confirmed the binding of C1INH and factor H to activated platelets. To this list we have added another potent complement inhibitor, C4BP. The binding of factor H and C4BP was shown to be dependent on exposure of CS-A on activated platelets. The physiological relevance of these reactions was reflected in an elevated expression of CD11b on leukocytes, and increased generation of platelet-leukocyte complexes. The platelets were involved in these events by at least two different mechanisms; generation of C5a which activated leukocytes and binding of C3(H2O)/iC3(H2O), a ligand to the intergrin CD11b/CD18 on their surface. These mechanisms add further to the understanding o, Platelet Mediated Complement Activation

Exposure of chondroitin sulfate A (CS-A) on the surface of activated platelets is well established. The aim of the present study was to investigate to what extent CS-A contributes to the binding of C1q and the complement regulators C1 inhibitor (C1INH), C4b-binding protein (C4BP), and factor H to platelets. Human serum was passed over Sepharose conjugated with CS-A, and bound proteins were identified by Western blotting, and mass spectrometric analysis. C1q was identified as the main protein that specifically bound to CS-A, but C4BP and factor H were also shown to interact. Binding of C1INH was dependent of the presence of C1q and not bound to CS-A from C1q-depleted serum. The specific interactions observed of these proteins with CS-A were subsequently confirmed by surface plasmon resonance analysis using purified proteins. Importantly, C1q, C4BP, and factor H were shown to bind also to activated platelets and this interaction was inhibited by a CS-A-specific monoclonal antibody, thereby linking the binding of C1q, C4BP, and factor H to exposure of CS-A on platelets. CS-A-bound C1q was also shown to amplify the binding of model immune complexes to both microtiter plate-bound CS-A and to activated platelets. In conclusion, this study supports the concept that CS-A contributes to the binding of C1q, C4BP, and factor H to platelets, thereby adding CS-A to the previously reported binding sites for these proteins on the platelet surface. CS-A-bound C1q seems to amplify the binding of immune complexes to activated platelets, suggesting a role for this molecule in immune complex diseases., platelet mediated complement activation

Blood platelets are emerging as important immunomodulatory cells, but complement interaction with platelets is not well understood. Several platelet structures have been described as complement protein 1q (C1q) binding receptors, such as C1qRp/CD93 and gC1qR. However, there are conflicting results whether these receptors are C1q binding structures, or even at all expressed on the cell surface. Recently, the collagen-binding integrin alphaIIbetaI was reported to bind C1q on mast cells, and this receptor is also present on platelets. The aim of this study was to further characterize the effects of C1q on platelets, by quantifying the platelet surface expression of P-selectin (CD62P) and monitoring the formation of platelet-neutrophil aggregates. Using flow cytometry, we found that C1q dose-dependently triggered a rapid but moderate and transient up-regulation of P-selectin already within 5s of C1q exposure. Pre-incubation with an antibody directed against gC1qR significantly inhibited (with 57% compared to control) the up-regulation, whereas an antibody towards the alphaIIbetaI-integrin showed no effect. Stimulation with C1q did not change the cytosolic calcium-levels, as measured with the fluorescent ratiometric probe Fura-2, however, a protein kinase C inhibitor (GF109203x) blocked the C1q-induced P-selectin expression. Furthermore, pre-incubation of platelets with C1q diminished both the collagen as well as the collagen-related peptide-induced up-regulation of P-selectin, most evident after 90s of stimulation. This indicates that C1q may regulate platelet activation via the GPVI receptor, which is a novel finding. Moreover, C1q antagonized the collagen-induced formation of platelet-neutrophil aggregates, indicating a reduced interaction between platelet P-selectin and neutrophil P-selectin glycoprotein ligand-1(PSGL-1/CD162). In summary, C1q induces a moderate rapid platelet P-selectin expression, modulates subsequent collagen and collagen-related peptide stimulati, Original Publication:Caroline Skoglund, Jonas Wetterö, Pentti Tengvall and Torbjörn Bengtsson, C1q induces a rapid up-regulation of P-selectin and modulates collagen- and collagen-related peptide-triggered activation in human platelets, 2010, Immunobiology, (215), 12, 987-995.http://dx.doi.org/10.1016/j.imbio.2009.11.004Copyright: Elsevier Science B. V., Amsterdamhttp://www.elsevier.com

OBJECTIVE: C1q deficiency is the strongest risk factor known for the development of systemic lupus erythematosus (SLE), since almost all humans with a genetic deficiency of C1q develop this disease. Low C1q serum concentration is also a typical finding in SLE during flares, emphasizing the involvement of C1q in SLE pathogenesis. Recent studies have revealed that C1q has a regulatory effect on Toll-like receptor-induced cytokine production. Therefore, we undertook this study to investigate whether C1q could regulate production of interferon-alpha (IFNalpha). METHODS: Peripheral blood mononuclear cells (PBMCs) and plasmacytoid dendritic cells (PDCs) were stimulated with 3 known interferogenic stimuli and cultured with physiologic concentrations of C1q. IFNalpha production was determined by an immunoassay. RESULTS: C1q significantly inhibited PBMC IFNalpha production induced by RNA-containing immune complexes (ICs), herpes simplex virus (HSV), and CpG DNA. C1q also inhibited PDC IFNalpha production induced by ICs and CpG DNA but increased PDC IFNalpha production induced by HSV. The regulatory role of C1q was not specific for IFNalpha but was also seen for interleukin-6 (IL-6), IL-8, and tumor necrosis factor alpha. We demonstrated binding of C1q to PDCs both by surface plasmon resonance interaction analysis and by flow cytometry, and we also demonstrated intracellular detection of 2 C1q binding proteins. CONCLUSION: Our findings contribute to the understanding of why C1q deficiency is such a strong risk factor for SLE and suggest an explanation for the up-regulation of the type I IFN system seen in SLE patients.

Prion protein (PrP) is an endogenous protein involved in the pathogenesis of bovine spongiform encephalopathy and Creutzfeldt–Jakob disease. Murine PrP has been reported to bind C1q and activate the classical pathway of complement in a copper-dependent manner. Here we show that various conformational isoforms (native, amyloid fibrils, and β-oligomers) of recombinant human PrP (90–231 and 121–231) bind C1q and activate complement. PrP binds both the globular head and collagenous stalk domains of C1q. Native, β-oligomeric and amyloid fibrils of PrP all activate the classical and alternative pathways of complement to different extent. However, they do not trigger the lectin pathway. Of the tested PrP conformational isoforms we find that β-oligomers bind C1q and activate complement most strongly. Membrane attack complex formation initiated by PrP is subdued in comparison to deposition of early complement components. This is most likely attributed to the interaction between human PrP and complement inhibitors factor H and C4b-binding protein. Accordingly, PrP-triggered complement activation in the terminal pathway was increased in serum lacking C4b-binding protein. Taken together the present study indicates that complement activation may be an important factor in human prion diseases, suggesting that complement induced activities may prove relevant therapeutic targets.

Udgivelsesdato: September

C-reactive protein (CRP) interacts with phosphorylcholine (PC), Fcγ receptors, complement factor C1q and cell nuclear constituents, yet its biological roles are insufficiently understood. The aim was to characterize CRP-induced complement activation by ellipsometry. PC conjugated with keyhole limpet hemocyanin (PC-KLH) was immobilized to cross-linked fibrinogen. A low-CRP serum with different amounts of added CRP was exposed to the PC-surfaces. The total serum protein deposition was quantified and deposition of IgG, C1q, C3c, C4, factor H, and CRP detected with polyclonal antibodies. The binding of serum CRP to PC-KLH dose-dependently triggered activation of the classical pathway. Unexpectedly, the activation was efficiently down-regulated at CRP levels >150 mg/L. Using radial immunodiffusion, CRP–C1q interaction was observed in serum samples with high CRP concentrations. We propose that the underlying mechanism depends on fluid-phase interaction between C1q and CRP. This might constitute another level of complement regulation, which has implications for systemic lupus erythematosus where CRP is often low despite flare-ups.

Trypanosoma cruzi calreticulin (TcCRT), described in our laboratory, retains several important functional features from its vertebrate homologues. We have shown that recombinant TcCRT inhibits the human complement system when it binds to the collagenous portion of C1q. The generation of classical pathway convertases and membrane attack complexes is thus strongly inhibited. In most T. cruzi-infected individuals, TcCRT is immunogenic and mediates the generation of specific antibodies. By reverting the C1q / TcCRT interaction, a parasite immune evasion strategy, these antibodies contribute to the host / parasite equilibrium. In an in vitro correlate of this situation, we show that the C1q / TcCRT interaction is inhibited by F(ab')2 polyclonal anti-TcCRT IgG fragments. It is therefore feasible that in infected humans anti-TcCRT antibodies participate in reverting an important parasite strategy aimed at inhibiting the classical complement pathway. Thus, membrane-bound TcCRT interacts with the collagenous portion C1q, and this C1q is recognized by the CD91-bound host cell CRT, thus facilitating parasite internalization. Based on our in vitro results, it could be proposed that the in vivo interaction between TcCRT and vertebrate C1q could be inhibited by F(ab')2 fragments anti-rTcCRT or against its S functional domain, thus interfering with the internalization process

Complement has been postulated to contribute to inflammatory reactions associated with the neuropathology of Alzheimer's disease (AD). C1q, an initial component of the complement cascade, is associated with neuritic plaques and with neurons in the hippocampus of AD brain. Here, we report the presence of C1q in a cognitively intact subject, previously identified as preclinical AD. We compared in detail brain tissue of this preclinical case with a genetically related late-onset AD case. In the AD brain, C1q was typically associated with fibrillar Abeta plaques in frontal cortex and with plaques and neurons in the hippocampus. In the preclinical subject, C1q was abundantly present but it was cell-associated only, being primarily colocalized with neurons in both frontal cortex and hippocampus. However, no predominant cortical neuronal C1q localization was found in other preclinical cases or in Down's cases of different ages. Thus, it is possible that this neuronal-associated C1q reflects an early, but transient, response to injury that may modulate the progression of neurological dysfunction in AD.