RESUMEN
Potency assessment of monoclonal antibodies or corresponding biosimilars in cell-based assays is an essential prerequisite in biopharmaceutical research and development. However, cellular bioassays are still subject to limitations in sample throughput, speed, and often need costly reagents or labels as they are based on an indirect readout by luminescence or fluorescence. In contrast, whole-cell Matrix-Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) Mass Spectrometry (MS) has emerged as a direct, fast and label-free technology for functional drug screening being able to unravel the molecular complexity of cellular response to pharmaceutical reagents. However, this approach has not yet been used for cellular testing of biologicals. In this study, we have conceived, developed and benchmarked a label-free MALDI-MS based cell bioassay workflow for the functional assessment of complement-dependent cytotoxicity (CDC) of Rituximab antibody. By computational evaluation of response profiles followed by subsequent m/z feature annotation via fragmentation analysis and trapped ion mobility MS, we identified adenosine triphosphate and glutathione as readily MS-assessable metabolite markers for CDC and demonstrate that robust concentration-response characteristics can be obtained by MALDI-TOF MS. Statistical assay performance indicators suggest that whole-cell MALDI-TOF MS could complement the toolbox for functional cellular testing of biopharmaceuticals.
Asunto(s)
Rituximab , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Humanos , Rituximab/farmacología , Proteínas del Sistema Complemento/metabolismo , Bioensayo/métodos , Anticuerpos Monoclonales , Glutatión/metabolismo , Adenosina Trifosfato/metabolismoRESUMEN
The complement system and neutrophils play crucial roles in innate immunity. Neutrophils release neutrophil extracellular traps (NETs), which are composed of decondensed DNA entangled with granular contents, as part of their innate immune function. Mechanisms governing complement-mediated NET formation remain unclear. In this study, we tested a two-step NETosis mechanism, as follows: classical complement-mediated neutrophil activation in serum and subsequent NET formation in serum-free conditions, using neutrophils from healthy donors, endothelial cells, and various assays (Fluo-4AM, DHR123, and SYTOX), along with flow cytometry and confocal microscopy. Our findings reveal that classical complement activation on neutrophils upregulated the membrane-anchored complement regulators CD46, CD55, and CD59. Additionally, complement activation increased CD11b on neutrophils, signifying activation and promoting their attachment to endothelial cells. Complement activation induced calcium influx and citrullination of histone 3 (CitH3) in neutrophils. However, CitH3 formation alone was insufficient for NET generation. Importantly, NET formation occurred only when neutrophils were in serum-free conditions. In such environments, neutrophils induced NADPH oxidase-dependent reactive oxygen species (ROS) production, leading to NET formation. Hence, we propose that complement-mediated NET formation involves a two-step process, as follows: complement deposition, neutrophil priming, calcium influx, CitH3 formation, and attachment to endothelial cells in serum. This is followed by NADPH-dependent ROS production and NET completion in serum-free conditions. Understanding this process may unveil treatment targets for pathologies involving complement activation and NET formation.
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Calcio , Activación de Complemento , Trampas Extracelulares , NADPH Oxidasas , Activación Neutrófila , Neutrófilos , Especies Reactivas de Oxígeno , Trampas Extracelulares/metabolismo , Humanos , Neutrófilos/metabolismo , Neutrófilos/inmunología , NADPH Oxidasas/metabolismo , Calcio/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Proteínas del Sistema Complemento/metabolismo , Células Endoteliales/metabolismo , Medio de Cultivo Libre de Suero/farmacología , Histonas/metabolismoRESUMEN
Venous thromboembolism (VTE) is a common, deadly disease with an increasing incidence despite preventive efforts. Clinical observations have associated elevated antibody concentrations or antibody-based therapies with thrombotic events. However, how antibodies contribute to thrombosis is unknown. Here, we show that reduced blood flow enabled immunoglobulin M (IgM) to bind to FcµR and the polymeric immunoglobulin receptor (pIgR), initiating endothelial activation and platelet recruitment. Subsequently, the procoagulant surface of activated platelets accommodated antigen- and FcγR-independent IgG deposition. This leads to classical complement activation, setting in motion a prothrombotic vicious circle. Key elements of this mechanism were present in humans in the setting of venous stasis as well as in the dysregulated immunothrombosis of COVID-19. This antibody-driven thrombosis can be prevented by pharmacologically targeting complement. Hence, our results uncover antibodies as previously unrecognized central regulators of thrombosis. These findings carry relevance for therapeutic application of antibodies and open innovative avenues to target thrombosis without compromising hemostasis.
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Plaquetas , COVID-19 , Activación de Complemento , Inmunoglobulina M , Trombosis , Humanos , Trombosis/inmunología , Animales , Inmunoglobulina M/inmunología , Activación de Complemento/inmunología , Ratones , Plaquetas/inmunología , Plaquetas/metabolismo , COVID-19/inmunología , COVID-19/complicaciones , SARS-CoV-2/inmunología , Proteínas del Sistema Complemento/inmunología , Proteínas del Sistema Complemento/metabolismo , Activación Plaquetaria/inmunología , Inmunoglobulina G/inmunología , MasculinoRESUMEN
The mammalian complement system constitutes a highly sophisticated body defense machinery. The evolutionary origin of the complement system can be traced to Coelenterata as the presence of the central component C3 and two activation proteases BF and MASP. In the present study, the main complement components were screened and analyzed from the genomes of different species in metazoan subphyla/phyla. C1q with classical domains can be traced to Annelida, and ficolin and MBL to Urochordata. C1r and C1s are only found in Chondrichthyes and even higher species, and MASP is traced to Coelenterata. In the evolutionary tree, C1r from Vertebrates is close to MASP1/2/3 from Deuterostomia and Coelenterata, and C1s from Vertebrates is close to MASP-like protease (MASPL) from Arthropoda, Mollusca, and Annelida. C2, BF, and DF can be traced to Mollusca, Coelenterata, and Porifera, respectively. There are no clear C2 and BF branches in the evolutionary tree. C3 can be traced to Coelenterata, and C4 and C5 are only in Chondrichthyes and even higher species. There are three clear C3, C4, and C5 branches in the evolutionary tree. C6-like (C6L) and C8 can be traced to Urochordata, and C7-like (C7L) can be traced to Cephalochordara. C6L, C7L, and C8 from Urochordata and Cephalochordara provide the structural conditions for the formation of Vertebrate MAC components. The findings unveil the evolutionary principles of the complement system and provide insight into its sophistication.
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Proteínas del Sistema Complemento , Evolución Molecular , Duplicación de Gen , Filogenia , Animales , Proteínas del Sistema Complemento/genética , Proteínas del Sistema Complemento/metabolismo , Serina Proteasas Asociadas a la Proteína de Unión a la Manosa/genética , Serina Proteasas Asociadas a la Proteína de Unión a la Manosa/metabolismo , Humanos , Complemento C3/genética , Complemento C3/metabolismo , Complemento C1s/metabolismo , Complemento C1s/genética , Complemento C1s/químicaRESUMEN
The complement (C) system is implicated in the etiopathogenesis of rheumatoid arthritis (RA). However, there is a lack of studies characterizing all three C pathways in RA patients. This study aimed to evaluate the association between an in-depth examination of the C system and RA patient characteristics, focusing on disease activity and the presence of rheumatoid factor and anti-citrullinated protein autoantibodies (ACPA). In a cohort of 430 RA patients, functional assays of the three C pathways (classical, alternative, and lectin) and serum levels of their components were assessed. Components included C1q (classical); factor D and properdin (alternative); lectin (lectin); C1-inhibitor; C2, C4, and C4b (classical and lectin); C3, C3a, and C4b (common); and C5, C5a, and C9 (terminal). A multivariable linear regression analysis showed significant positive correlations between C-reactive protein and C system proteins and functional assays, especially in the terminal and common pathways. Disease activity, measured by scores with or without acute phase reactants, positively correlated with the classical pathway functional test and terminal pathway products. Conversely, rheumatoid factor or ACPA presence was associated with lower classical pathway values and decreased C3a and C4b levels, suggesting complement depletion. In conclusion, RA disease activity increases C molecules and functional complement assays, while rheumatoid factor or ACPA positivity is linked to C consumption. Our study offers a detailed analysis of the complement system's role in RA, potentially guiding the development of more targeted and effective treatment strategies.
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Artritis Reumatoide , Factor Reumatoide , Humanos , Artritis Reumatoide/sangre , Artritis Reumatoide/inmunología , Artritis Reumatoide/metabolismo , Masculino , Femenino , Persona de Mediana Edad , Anciano , Factor Reumatoide/sangre , Adulto , Proteínas del Sistema Complemento/metabolismo , Proteínas del Sistema Complemento/inmunología , Anticuerpos Antiproteína Citrulinada/sangre , Vía Alternativa del Complemento , Activación de Complemento , Proteína C-Reactiva/metabolismo , Proteína C-Reactiva/análisis , Vía Clásica del ComplementoRESUMEN
Cancer-specific monoclonal antibodies (CasMabs) that recognize cancer-specific antigens with in vivo antitumor efficacy are innovative therapeutic strategies for minimizing adverse effects. We previously established a cancer-specific anti-human epidermal growth factor receptor 2 (HER2) monoclonal antibody (mAb), H2Mab-250/H2CasMab-2. In flow cytometry and immunohistochemistry, H2Mab-250 reacted with HER2-positive breast cancer cells but did not show reactivity to normal epithelial cells. In contrast, a clinically approved anti-HER2 mAb, trastuzumab, strongly recognizes both breast cancer and normal epithelial cells in flow cytometry. The human IgG1 version of H2Mab-250 (H2Mab-250-hG1) possesses compatible in vivo antitumor effects against breast cancer xenografts to trastuzumab despite the lower affinity and effector activation than trastuzumab in vitro. This study compared the antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cellular cytotoxicity (CDC) between H2Mab-250-hG1 and trastuzumab. Both H2Mab-250-hG1 and trastuzumab showed ADCC activity against HER2-overexpressed Chinese hamster ovary -K1 and breast cancer cell lines (BT-474 and SK-BR-3) in the presence of human natural killer cells. Some tendency was observed where trastuzumab showed a more significant ADCC effect compared to H2Mab-250-hG1. Importantly, H2Mab-250-hG1 exhibited superior CDC activity in these cells compared to trastuzumab. Similar results were obtained in the mouse IgG2a types of both H2Mab-250 and trastuzumab. These results suggest the different contributions of ADCC and CDC activities to the antitumor effects of H2Mab-250-hG1 and trastuzumab, and indicate a future direction for the clinical development of H2Mab-250-hG1 against HER2-positive tumors.
Asunto(s)
Citotoxicidad Celular Dependiente de Anticuerpos , Cricetulus , Receptor ErbB-2 , Trastuzumab , Trastuzumab/farmacología , Animales , Humanos , Receptor ErbB-2/metabolismo , Receptor ErbB-2/antagonistas & inhibidores , Receptor ErbB-2/inmunología , Citotoxicidad Celular Dependiente de Anticuerpos/efectos de los fármacos , Células CHO , Línea Celular Tumoral , Femenino , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/inmunología , Neoplasias de la Mama/patología , Antineoplásicos Inmunológicos/farmacología , Anticuerpos Monoclonales/farmacología , Proteínas del Sistema Complemento/metabolismo , Proteínas del Sistema Complemento/inmunología , Ratones , CricetinaeRESUMEN
Background: Studies comparing cerebrospinal fluid (CSF) and plasma complement proteins in Alzheimer's disease (AD) patients versus healthy controls (HC) have yielded inconsistent results. Discrepancies in the preanalytical sample handling could contribute to the heterogeneity in the reported findings. Objective: Using qualified immunoassays, we aimed at assessing the impact of preanalytical procedures on complement proteins in blood and CSF from AD patients and HCs. Methods: We supplemented HC and AD CSF/plasma with complement stabilizers and measured the complement proteins C4a, C4, C3a, C3, Factor Bb and Factor B by immunoassay. We tested the impact of freeze-thaw (FT) cycles on fluid complement proteins. Results: Most complement proteins were mildly impacted by FT cycles in plasma but not CSF, except for C3a which displayed greater sensitivity to FTs in CSF than in plasma. In CSF, the effect of FTs on C3a was reduced but not prevented by the supplementation with EDTA (±Futhan). Conclusions: Our findings provide recommendations for CSF/plasma sample handling to ensure robust and reproducible complement biomarker analyses in AD.
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Enfermedad de Alzheimer , Biomarcadores , Proteínas del Sistema Complemento , Humanos , Enfermedad de Alzheimer/sangre , Enfermedad de Alzheimer/líquido cefalorraquídeo , Enfermedad de Alzheimer/diagnóstico , Masculino , Femenino , Anciano , Biomarcadores/sangre , Biomarcadores/líquido cefalorraquídeo , Proteínas del Sistema Complemento/metabolismo , Persona de Mediana Edad , Manejo de Especímenes , Inmunoensayo/métodos , Anciano de 80 o más AñosRESUMEN
Knowledge about the molecular mechanisms underlying the systemic inflammation observed in psoriasis remains incomplete. In this study, we applied mass spectrometry-based proteomics to compare the plasma protein levels between patients with psoriasis and healthy individuals, aiming to unveil potential systemically dysregulated proteins and pathways associated with the disease. Plasma samples from adult patients with moderate-to-severe psoriasis vulgaris (N = 59) and healthy age- and sex-matched individuals (N = 21) were analyzed using liquid chromatography-tandem mass spectrometry. Patients did not receive systemic anti-psoriatic treatment for four weeks before inclusion. A total of 776 protein groups were quantified. Of these, 691 were present in at least 60% of the samples, providing the basis for the downstream analysis. We identified 20 upregulated and 22 downregulated proteins in patients with psoriasis compared to controls (p < 0.05). Multiple proteins from the complement system were upregulated, including C2, C4b, C5, and C9, and pathway analysis revealed enrichment of proteins involved in complement activation and formation of the terminal complement complex. On the other end of the spectrum, periostin was the most downregulated protein in sera from patients with psoriasis. This comprehensive proteomic investigation revealed significantly elevated levels of complement cascade proteins in psoriatic plasma, which might contribute to increased systemic inflammation in patients with psoriasis.
Asunto(s)
Proteínas del Sistema Complemento , Proteómica , Psoriasis , Humanos , Psoriasis/sangre , Psoriasis/metabolismo , Masculino , Femenino , Proteómica/métodos , Adulto , Proteínas del Sistema Complemento/metabolismo , Persona de Mediana Edad , Estudios de Casos y Controles , Espectrometría de Masas en Tándem , Proteoma/metabolismo , Biomarcadores/sangre , Cromatografía LiquidaRESUMEN
Autoimmune related kidney diseases (ARKDs), including minimal change nephropathy (MCN), membranous nephropathy (MN), IgA nephropathy (IgAN), and lupus nephritis (LN), significantly affect renal function. These diseases are characterized by the formation of local immune complexes and the subsequent activation of the complement system, leading to kidney damage and proteinuria. Despite the known patterns of glomerular injury, the specific molecular mechanisms that contribute to renal tubular damage across ARKDs remain underexplored. Laser capture microdissection and liquid chromatography-tandem mass spectrometry (LC-MS/MS) were used to conduct a comparative proteomic analysis of renal tubular tissues from formalin-fixed paraffin-embedded samples. The cohort comprised of 10 normal controls (NC), 5 MCN, 4 MN, 17 IgAN, and 21 LN patients. Clinical parameters and histopathological assessments were integrated with proteomic findings to comprehensively investigate underlying pathogenic processes. Clinical evaluation indicated significant glomerular damage, as reflected by elevated urinary protein levels and reduced plasma albumin levels in patients with ARKD. Histological analyses confirmed varying degrees of tubular damage and deposition of immune complexes. Proteomic analyses identified significant changes in protein expression, particularly in complement components (C3, C4A, C4B, C8G, CFB, and SERPINA1) and mitochondrial proteins (ATP5F1E and ATP5PD), highlighting the common alterations in the complement system and mitochondrial proteins across ARKDs. These alterations suggest a novel complement-mitochondrial-epithelial-mesenchymal transition (EMT) pathway axis that contributes to tubular damage in ARKDs. Notably, significant alterations in CFB in tubular ARKD patients were revealed, implicating it as a therapeutic target. This study underscores the importance of complement activation and mitochondrial dysfunction in the pathogenesis of ARKDs, and proposes CFB as a potential therapeutic target to inhibit complement activation and mitigate tubular damage. Future research should validate the complement-mitochondrial-EMT pathway axis and explore the effects and mechanisms of CFB inhibitors in alleviating ARKD progression.
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Activación de Complemento , Mitocondrias , Proteómica , Humanos , Proteómica/métodos , Femenino , Masculino , Adulto , Mitocondrias/metabolismo , Persona de Mediana Edad , Enfermedades Renales/metabolismo , Enfermedades Renales/patología , Enfermedades Renales/inmunología , Enfermedades Autoinmunes/metabolismo , Enfermedades Autoinmunes/inmunología , Enfermedades Autoinmunes/patología , Túbulos Renales/metabolismo , Túbulos Renales/patología , Espectrometría de Masas en Tándem , Captura por Microdisección con Láser , Proteínas del Sistema Complemento/metabolismo , Cromatografía LiquidaRESUMEN
Complement and extracellular vesicles (EVs) association with thrombogenic tendencies is acknowledged, but limited evidence exists for their link to COVID-19 venous thromboembolism. This study aims to examine the relationship between pulmonary embolism and the expression of complement and other proteins related to thrombogenesis in severe Covid-19 patients. We included prospectively 207 severe COVID-19 patients and retrospectively screened for pulmonary embolism (PE). This analysis comprises 20 confirmed PE cases and 20 matched patients without PE. Blood samples taken at the admission in the intensive care unit were analyzed for complement using ELISA. EVs derived from neutrophils, endothelium, or platelets, as well carrying complement or tissue factor were analyzed using flow cytometry. Complement levels were markedly elevated, with a notable increase in C3a and Terminal Complement Complex. The most prevalent EV population was identified as tissue factor (TF)-carrying EVs which peaked in patients with PE during ICU days 4-9. However, for both the complement and analyzed EV populations, no statistically significant differences were found between the patients who developed pulmonary embolism and those who did not. In conclusion, complement factors and EVs expressing tissue factor, along with EVs derived from endothelial cells and platelets, are elevated in severe COVID-19 patients, regardless of the presence of pulmonary embolism. However, the involvement of complement and procoagulant EVs in peripheral plasma in the development of pulmonary embolism is still unclear and requires further investigation.
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COVID-19 , Proteínas del Sistema Complemento , Vesículas Extracelulares , Embolia Pulmonar , SARS-CoV-2 , Humanos , Embolia Pulmonar/sangre , COVID-19/complicaciones , COVID-19/sangre , Vesículas Extracelulares/metabolismo , Masculino , Femenino , Persona de Mediana Edad , Anciano , Proteínas del Sistema Complemento/metabolismo , Estudios Retrospectivos , Plaquetas/metabolismo , Plaquetas/patología , Tromboplastina/metabolismo , Adulto , Estudios ProspectivosRESUMEN
Alzheimer's Disease (AD) is a neurodegenerative disease characterized by profound memory impairments, synaptic loss, neuroinflammation, and hallmark pathological markers. High-fat diet (HFD) consumption increases the risk of developing AD even after controlling for metabolic syndrome, pointing to a role of the diet itself in increasing risk. In AD, the complement system, an arm of the immune system which normally tags redundant or damaged synapses for pruning, becomes pathologically overactivated leading to tagging of healthy synapses. While the unhealthy diet to AD link is strong, the underlying mechanisms are not well understood in part due to confounding variables associated with long-term HFD which can independently influence the brain. Therefore, we experimented with a short-term diet regimen to isolate the diet's impact on brain function without causing obesity. This project investigated the effect of short-term HFD on 1) memory, 2) neuroinflammation including complement, 3) AD pathology markers, 4) synaptic markers, and 5) in vitro microglial synaptic phagocytosis in the 3xTg-AD mouse model. Following the consumption of either standard chow or HFD, 3xTg-AD and non-Tg mice were tested for memory impairments. In a separate cohort of mice, levels of hippocampal inflammatory markers, complement proteins, AD pathology markers, and synaptic markers were measured. For the last set of experiments, BV2 microglial phagocytosis of synapses was evaluated. Synaptoneurosomes isolated from the hippocampus of 3xTg-AD mice fed chow or HFD were incubated with equal numbers of BV2 microglia. The number of BV2 microglia that phagocytosed synaptoneurosomes was tracked over time with a live-cell imaging assay. Finally, we incubated BV2 microglia with a complement receptor inhibitor (NIF) and repeated the assay. Behavioral analysis showed 3xTg-AD mice had significantly impaired long-term contextual and cued fear memory compared to non-Tg mice that was further impaired by HFD. HFD significantly increased inflammatory markers and complement expression while decreasing synaptic marker expression only in 3xTg-AD mice, without altering AD pathology markers. Synaptoneurosomes from HFD-fed 3xTg-AD mice were phagocytosed at a significantly higher rate than those from chow-fed mice, suggesting the synapses were altered by HFD. The complement receptor inhibitor blocked this effect in a dose-dependent manner, demonstrating the HFD-mediated increase in phagocytosis was complement dependent. This study indicates HFD consumption increases neuroinflammation and over-activates the complement cascade in 3xTg-AD mice, resulting in poorer memory. The in vitro data point to complement as a potential mechanistic culprit and therapeutic target underlying HFD's influence in increasing cognitive vulnerability to AD.
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Enfermedad de Alzheimer , Dieta Alta en Grasa , Modelos Animales de Enfermedad , Ratones Transgénicos , Microglía , Sinapsis , Animales , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/inmunología , Dieta Alta en Grasa/efectos adversos , Ratones , Sinapsis/metabolismo , Microglía/metabolismo , Trastornos de la Memoria/metabolismo , Trastornos de la Memoria/inmunología , Masculino , Proteínas del Sistema Complemento/metabolismo , Memoria/fisiología , Enfermedades Neuroinflamatorias/metabolismo , Enfermedades Neuroinflamatorias/inmunología , Ratones Endogámicos C57BL , Encéfalo/metabolismo , Encéfalo/inmunología , Hipocampo/metabolismo , Neuroinmunomodulación/fisiologíaRESUMEN
The plasma bacterial killing ability (BKA) is modulated by the stress response in vertebrates, including amphibians. The complement system is an effector mechanism comprised of a set of proteins present in the plasma that once activated can promote bacterial lysis. Herein, we investigated whether changes in plasma BKA as a result of the acute stress response and an immune challenge are mediated by the complement system in Rhinella diptycha toads. Additionally, we investigated whether the observed changes in plasma BKA are associated with changes in plasma corticosterone levels (CORT). We subjected adult male toads to a restraint or an immune challenge (with three concentrations of Aeromonas hydrophila heat inactivated), and then evaluated the plasma BKA against A. hydrophila, in vitro. We determined the complement system activity on plasma BKA, by treating the plasma (baseline, 1 h and 24 h post-restraint, and after the immune challenge) with ethylenediaminetetraacetic acid, heat, or protease. Our results showed increased CORT 1 h and 24 h after restraint and decreased plasma BKA 24 h post-restraint. The inhibitors of the complement system decreased the plasma BKA compared with untreated plasma at all times (baseline, 1 h, and 24 h after restraint), demonstrating that the plasma BKA activity is partially mediated by the complement system. The immune challenge increased CORT, with the highest values being observed in the highest bacterial concentration, compared with control. The plasma BKA was not affected by the immune challenge but was demonstrated to be partially mediated by the complement system. Our results demonstrated that restraint and the immune challenge activated the hypothalamus-pituitary-interrenal axis, by increasing plasma CORT levels in R. diptycha. Also, our results demonstrated the complement system is participative in the plasma BKA for baseline and post-stress situations in these toads.
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Aeromonas hydrophila , Proteínas del Sistema Complemento , Corticosterona , Estrés Fisiológico , Animales , Proteínas del Sistema Complemento/metabolismo , Proteínas del Sistema Complemento/inmunología , Masculino , Estrés Fisiológico/inmunología , Aeromonas hydrophila/fisiología , Aeromonas hydrophila/inmunología , Corticosterona/sangre , Actividad Bactericida de la Sangre , Bufonidae/inmunología , Bufonidae/microbiología , Bufonidae/sangreRESUMEN
Antibodies to Ebola virus glycoprotein (EBOV GP) represent an important correlate of the vaccine efficiency and infection survival. Both neutralization and some of the Fc-mediated effects are known to contribute the protection conferred by antibodies of various epitope specificities. At the same time, the role of the complement system remains unclear. Here, we compare complement activation by two groups of representative monoclonal antibodies (mAbs) interacting with the glycan cap (GC) or the membrane-proximal external region (MPER) of GP. Binding of GC-specific mAbs to GP induces complement-dependent cytotoxicity (CDC) in the GP-expressing cell line via C3 deposition on GP in contrast to MPER-specific mAbs. In the mouse model of EBOV infection, depletion of the complement system leads to an impairment of protection exerted by one of the GC-specific, but not MPER-specific mAbs. Our data suggest that activation of the complement system represents an important mechanism of antiviral protection by GC antibodies.
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Anticuerpos Monoclonales , Anticuerpos Antivirales , Ebolavirus , Fiebre Hemorrágica Ebola , Polisacáridos , Proteínas del Envoltorio Viral , Animales , Ebolavirus/inmunología , Anticuerpos Monoclonales/inmunología , Ratones , Fiebre Hemorrágica Ebola/inmunología , Fiebre Hemorrágica Ebola/virología , Fiebre Hemorrágica Ebola/prevención & control , Polisacáridos/inmunología , Anticuerpos Antivirales/inmunología , Humanos , Proteínas del Envoltorio Viral/inmunología , Proteínas del Envoltorio Viral/metabolismo , Activación de Complemento , Ratones Endogámicos BALB C , Femenino , Proteínas del Sistema Complemento/inmunología , Proteínas del Sistema Complemento/metabolismo , Glicoproteínas/inmunologíaRESUMEN
BACKGROUND: Patients with rheumatoid arthritis (RA) have an increased risk of cardiovascular (CV) events and CV mortality. Subclinical carotid atherosclerosis is independently associated with rates of incident CV events among patients with RA. The complement system has been related to both the etiopathogenesis of RA and CV disease. In this study, we aimed to evaluate the association between a comprehensive assessment of the complement system and carotid intima media thickness and carotid plaque in patients with RA. METHODS: 430 patients with RA were recruited. Functional assays of the three pathways of the complement system, utilizing new-generation techniques, were assessed. Additionally, serum levels of individual components of the complement system belonging to the three pathways were measured: C1q (classical), lectin (lectin), C2, C4, and C4b (classical and lectin), factor D and properdin (alternative), C3 and C3a (common), C5, C5a, and C9 (terminal), as well as regulators factor I and C1-inhibitor. Subclinical carotid atherosclerosis was evaluated by ultrasonography. Multivariable linear regression analysis was conducted to investigate the association between the complement system and carotid intima media thickness and carotid plaque. RESULTS: After multivariable adjustment, which included traditional CV risk factors and disease-related data, C3a and C5a exhibited significant positive correlations with carotid intima media thickness. Additionally, higher values of C1-inhibitor, properdin, C3, C5, and C5a were independently associated with the presence of carotid plaque. CONCLUSION: The complement system and subclinical carotid atherosclerosis are linked in patients with RA.
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Artritis Reumatoide , Enfermedades de las Arterias Carótidas , Grosor Intima-Media Carotídeo , Humanos , Masculino , Artritis Reumatoide/sangre , Artritis Reumatoide/complicaciones , Femenino , Enfermedades de las Arterias Carótidas/sangre , Enfermedades de las Arterias Carótidas/epidemiología , Enfermedades de las Arterias Carótidas/diagnóstico por imagen , Persona de Mediana Edad , Anciano , Proteínas del Sistema Complemento/metabolismo , Proteínas del Sistema Complemento/análisis , Adulto , Estudios TransversalesRESUMEN
The complement system (C) is a crucial component of the innate immune system. An increasing body of research has progressively shed light on the pivotal role of C in immunological tolerance at the feto-maternal interface. Excessive C activation or impaired C regulation may determine the onset of pregnancy-related pathological conditions, including pre-eclampsia (PE). Thus, several studies have investigated the presence of C components or split products in blood matrixes (i.e., plasma, serum), urine, and amniotic fluid in PE. In the current study, we systematically reviewed the currently available scientific literature reporting measurements of C components as circulating biomarkers in PE, based on a literature search using Pubmed, Scopus, and Embase databases. A total of 41 out of 456 studies were selected after full-text analysis. Fourteen studies (34.1%) were identified as measuring the blood concentrations of the classical pathway, 5 (12.1%) for the lectin pathway, 28 (68.3%) for the alternative pathway, 17 (41.5%) for the terminal pathway components, and 16 (39%) for C regulators. Retrieved results consistently reported C4, C3, and factor H reduction, and increased circulating levels of C4d, Bb, factor D, C3a, C5a, and C5b-9 in PE compared to normal pregnancies, depicting an overall scenario of excessive C activation and aberrant C regulation. With evidence of C activation and dysregulation, C-targeted therapy is an intriguing perspective in PE management. Moreover, we also discussed emerging pitfalls in C analysis, mainly due to a lack of experimental uniformity and biased cohort selection among different studies and laboratories, aiming to raise a more comprehensive awareness for future standardization. Systematic review registration: https://www.crd.york.ac.uk/prospero/, identifier CRD42024503070.
Asunto(s)
Biomarcadores , Proteínas del Sistema Complemento , Preeclampsia , Humanos , Preeclampsia/sangre , Preeclampsia/inmunología , Preeclampsia/diagnóstico , Embarazo , Biomarcadores/sangre , Femenino , Proteínas del Sistema Complemento/metabolismo , Proteínas del Sistema Complemento/inmunología , Proteínas del Sistema Complemento/análisis , Activación de ComplementoRESUMEN
The complement and coagulation systems are ancestrally related mechanisms of serine protease-induced protein activation. Recent studies have shown that the complement system enhances platelet aggregation by activating platelets and vascular endothelial cells. This system is also involved in the expression of tissue factor, which induces the coagulation reaction. Activated platelets and coagulation factors are also known to activate the complement system. In diseases involving the complement system, such as paroxysmal nocturnal hemoglobinuria, autoimmune hemolytic anemia, and atypical hemolytic uremic syndrome, excessive activation of this system contributes to complement-mediated thrombosis. The anti-C5 antibody eculizumab has shown a remarkable thromboprophylactic effect in these complement diseases. The recent surge in development of new anti-complement agents has raised expectations for the advancement of treatments and preventive measures for thrombosis associated with complement disorders. This review outlines the crosstalk between these two systems, and describes the mechanisms of several diseases featuring both thrombosis and complement activation.
Asunto(s)
Coagulación Sanguínea , Activación de Complemento , Proteínas del Sistema Complemento , Humanos , Proteínas del Sistema Complemento/metabolismo , Trombosis , AnimalesRESUMEN
Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a debilitating chronic condition that is characterized by unresolved fatigue, post-exertion symptom exacerbation (PESE), cognitive dysfunction, orthostatic intolerance, and other symptoms. ME/CFS lacks established clinical biomarkers and requires further elucidation of disease mechanisms. A growing number of studies demonstrate signs of hematological and cardiovascular pathology in ME/CFS cohorts, including hyperactivated platelets, endothelial dysfunction, vascular dysregulation, and anomalous clotting processes. To build on these findings, and to identify potential biomarkers that can be related to pathophysiology, we measured differences in protein expression in platelet-poor plasma (PPP) samples from 15 ME/CFS study participants and 10 controls not previously infected with SARS-CoV-2, using DIA LC-MS/MS. We identified 24 proteins that are significantly increased in the ME/CFS group compared to the controls, and 21 proteins that are significantly downregulated. Proteins related to clotting processes - thrombospondin-1 (important in platelet activation), platelet factor 4, and protein S - were differentially expressed in the ME/CFS group, suggestive of a dysregulated coagulation system and abnormal endothelial function. Complement machinery was also significantly downregulated, including C9 which forms part of the membrane attack complex. Additionally, we identified a significant upregulation of lactotransferrin, protein S100-A9, and an immunoglobulin variant. The findings from this experiment further implicate the coagulation and immune system in ME/CFS, and bring to attention the pathology of or imposed on the endothelium. This study highlights potential systems and proteins that require further research with regards to their contribution to the pathogenesis of ME/CFS, symptom manifestation, and biomarker potential, and also gives insight into the hematological and cardiovascular risk for ME/CFS individuals affected by diabetes mellitus.
Asunto(s)
Biomarcadores , Coagulación Sanguínea , Regulación hacia Abajo , Síndrome de Fatiga Crónica , Espectrometría de Masas en Tándem , Humanos , Masculino , Femenino , Persona de Mediana Edad , Adulto , Cromatografía Liquida , Biomarcadores/sangre , Síndrome de Fatiga Crónica/sangre , Síndrome de Fatiga Crónica/fisiopatología , Síndrome de Fatiga Crónica/inmunología , Síndrome de Fatiga Crónica/metabolismo , Estudios de Casos y Controles , Proteómica , COVID-19/sangre , Proteínas del Sistema Complemento/metabolismo , Endotelio Vascular/metabolismo , Endotelio Vascular/fisiopatología , Cromatografía Líquida con Espectrometría de MasasRESUMEN
The complement system, composed of complement components and complement control proteins, plays an essential role in innate immunity. Complement system molecules are expressed at the maternal-conceptus interface, and inappropriate activation of the complement system is associated with various adverse pregnancy outcomes in humans and rodents. However, the expression, regulation, and function of the complement system at the maternal-conceptus interface in pigs have not been studied. In this study, we investigated the expression, localization, and regulation of complement system molecules at the maternal-conceptus interface in pigs. Complement components and complement control proteins were expressed in the endometrium, early-stage conceptus, and chorioallantoic tissues during pregnancy. The expression of complement components acting on the early stage of complement activation increased in the endometrium on Day 15 of pregnancy, with greater levels on that day compared with the estrous cycle. Localization of several complement components and complement control proteins was cell-type specific in the endometrium. The expression of C1QC, C2, C3, C4A, CFI, ITGB2, MASP1, and SERPING1 was increased by IFNG in endometrial explant tissues. Furthermore, cleaved C3 fragments were detected in endometrial tissues and uterine flushings on Day 15 of the estrous cycle and Day 15 of pregnancy, with greater levels on Day 15 of pregnancy. These results suggest that complement system molecules in pigs expressed at the maternal-conceptus interface play important roles in the establishment and maintenance of pregnancy by regulating innate immunity and modulating the maternal immune environment during pregnancy.
Asunto(s)
Activación de Complemento , Proteínas del Sistema Complemento , Endometrio , Animales , Femenino , Embarazo , Proteínas del Sistema Complemento/inmunología , Proteínas del Sistema Complemento/metabolismo , Endometrio/inmunología , Endometrio/metabolismo , Porcinos/inmunología , Activación de Complemento/inmunología , Inmunidad Innata , Membrana Corioalantoides/metabolismo , Membrana Corioalantoides/inmunologíaRESUMEN
Background: The complement system has been linked to the etiopathogenesis of rheumatoid arthritis (RA). Patients with RA exhibit a dysregulated profile of lipid molecules, which has been attributed to the inflammation present in the disease. In this study, we aimed to evaluate the association between a comprehensive assessment of the complement system and the lipid profile of patients with RA. Methods: 430 patients with RA were recruited. New-generation techniques were employed to conduct functional assays of the three pathways of the complement system. Serum levels of various complement components such as C1q, factor D, properdin, lectin, C1-inhibitor, C2, C4, C4b, C3, C3a, C5, C5a, and C9 were assessed. Furthermore, a complete pattern of lipid molecules was measured including high (HDL), low-density lipoproteins (LDL), and lipoprotein (a). Multivariable linear regression analysis was conducted to investigate the association between the complement system and lipid profile in RA patients. Results: After multivariable analysis, several noteworthy associations emerged between the complement system and lipid molecules. Notably, complement components most strongly linked to the lipid profile were C1q and properdin, representing the upstream classical and alternative pathways, along with C3 from the common cascade. These associations demonstrated significance and positivity concerning total cholesterol, LDL, atherogenic index, apolipoprotein B, and lipoprotein(a), suggesting a connection with an unfavorable lipid profile. Interestingly, complement functional assays of the three pathways and activated products such as C3a and C5a showed no correlation with the lipid pattern. Conclusion: The correlation between the complement system and lipid molecule patterns is pronounced in patients with RA. This relationship is predominantly positive and primarily associated with upstream complement components rather than activated ones.
Asunto(s)
Artritis Reumatoide , Proteínas del Sistema Complemento , Lípidos , Humanos , Artritis Reumatoide/sangre , Artritis Reumatoide/inmunología , Femenino , Masculino , Persona de Mediana Edad , Lípidos/sangre , Proteínas del Sistema Complemento/metabolismo , Proteínas del Sistema Complemento/inmunología , Adulto , Anciano , Activación de Complemento , Biomarcadores/sangre , Complemento C1q/metabolismo , Complemento C1q/inmunologíaRESUMEN
Thrombotic microangiopathy (TMA) is a pathological lesion that occurs due to endothelial injury. It can be seen in a heterogenous group of disorders, typically characterized by microangiopathic hemolytic anemia, thrombocytopenia, and end-organ ischemia. TMA can also be renal limited with no systemic manifestations. There are multiple etiologies of a TMA with complement activation being a core underlying mechanism, although the nature and extent of complement involvement can vary. A further complicated factor is the cross talk between complement, neutrophils, and coagulation pathways in the pathophysiology of TMAs. Therefore, a thorough and systematic clinical history and laboratory evaluation are critical to establish the cause and pathophysiology of a TMA. Furthermore, TMAs are associated with significant morbidity and mortality, and timely diagnosis is key for appropriate management and to prevent end-stage kidney disease and other associated complications. In this review, we focus on the pathology, mechanisms, diagnostic work up and treatment of TMAs associated with various etiologies. We also define the complement evaluations that should be conducted in these patients and further highlight the currently approved complement therapies as well as others in the pipeline.