RESUMEN
Extremely rare reactions characterized by thrombosis and thrombocytopenia have been described in subjects that received ChAdOx1 nCoV-19 vaccination 5-16 days earlier. Although patients with vaccine-induced thrombotic thrombocytopenia (VITT) have high levels of antibodies to platelet factor 4 (PF4)-polyanion complexes, the exact mechanism of the development of thrombosis is still unknown. Here we reported serum studies as well as proteomics and genomics analyses demonstrating a massive complement activation potentially linked to the presence of anti-PF4 antibodies in a patient with severe VITT. At admission, complement activity of the classical and lectin pathways were absent (0% for both) with normal levels of the alternative pathway (73%) in association with elevated levels of the complement activation marker sC5b-9 (630 ng/mL [n.v. 139-462 ng/mL]) and anti-PF4 IgG (1.918 OD [n.v. 0.136-0.300 OD]). The immunoblotting analysis of C2 showed the complete disappearance of its normal band at 110 kDa. Intravenous immunoglobulin treatment allowed to recover complement activity of the classical pathway (91%) and lectin pathway (115%), to reduce levels of sC5b-9 (135 ng/mL) and anti-PF4 IgG (0.681 OD) and to normalize the C2 pattern at immunoblotting. Proteomics and genomics analyses in addition to serum studies showed that the absence of complement activity during VITT was not linked to alterations of the C2 gene but rather to a strong complement activation leading to C2 consumption. Our data in a single patient suggest monitoring complement parameters in other VITT patients considering also the possibility to target complement activation with specific drugs.
Asunto(s)
Vacunas contra la COVID-19/efectos adversos , COVID-19/prevención & control , Complemento C2 , Complejo de Ataque a Membrana del Sistema Complemento , Vía Clásica del Complemento , Lectina de Unión a Manosa de la Vía del Complemento , Púrpura Trombocitopénica Trombótica , SARS-CoV-2 , Adulto , Autoanticuerpos/sangre , Vacunas contra la COVID-19/administración & dosificación , ChAdOx1 nCoV-19 , Complemento C2/genética , Complemento C2/metabolismo , Complejo de Ataque a Membrana del Sistema Complemento/genética , Complejo de Ataque a Membrana del Sistema Complemento/metabolismo , Vía Clásica del Complemento/efectos de los fármacos , Vía Clásica del Complemento/genética , Lectina de Unión a Manosa de la Vía del Complemento/efectos de los fármacos , Lectina de Unión a Manosa de la Vía del Complemento/genética , Femenino , Humanos , Factor Plaquetario 4/sangre , Púrpura Trombocitopénica Trombótica/sangre , Púrpura Trombocitopénica Trombótica/inducido químicamente , Púrpura Trombocitopénica Trombótica/genéticaRESUMEN
In COVID-19, acute respiratory distress syndrome (ARDS) and thrombotic events are frequent, life-threatening complications. Autopsies commonly show arterial thrombosis and severe endothelial damage. Endothelial damage, which can play an early and central pathogenic role in ARDS and thrombosis, activates the lectin pathway of complement. Mannan-binding lectin-associated serine protease-2 (MASP-2), the lectin pathway's effector enzyme, binds the nucleocapsid protein of severe acute respiratory syndrome-associated coronavirus-2 (SARS-CoV-2), resulting in complement activation and lung injury. Narsoplimab, a fully human immunoglobulin gamma 4 (IgG4) monoclonal antibody against MASP-2, inhibits lectin pathway activation and has anticoagulant effects. In this study, the first time a lectin-pathway inhibitor was used to treat COVID-19, six COVID-19 patients with ARDS requiring continuous positive airway pressure (CPAP) or intubation received narsoplimab under compassionate use. At baseline and during treatment, circulating endothelial cell (CEC) counts and serum levels of interleukin-6 (IL-6), interleukin-8 (IL-8), C-reactive protein (CRP) and lactate dehydrogenase (LDH) were assessed. Narsoplimab treatment was associated with rapid and sustained reduction of CEC and concurrent reduction of serum IL-6, IL-8, CRP and LDH. Narsoplimab was well tolerated; no adverse drug reactions were reported. Two control groups were used for retrospective comparison, both showing significantly higher mortality than the narsoplimab-treated group. All narsoplimab-treated patients recovered and survived. Narsoplimab may be an effective treatment for COVID-19 by reducing COVID-19-related endothelial cell damage and the resultant inflammation and thrombotic risk.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , COVID-19/inmunología , Lectina de Unión a Manosa de la Vía del Complemento/efectos de los fármacos , Endotelio Vascular/efectos de los fármacos , SARS-CoV-2/inmunología , Microangiopatías Trombóticas/tratamiento farmacológico , Anticuerpos Monoclonales/inmunología , Proteína C-Reactiva/inmunología , Proteína C-Reactiva/metabolismo , COVID-19/complicaciones , COVID-19/virología , Lectina de Unión a Manosa de la Vía del Complemento/inmunología , Endotelio Vascular/inmunología , Endotelio Vascular/patología , Femenino , Humanos , Inmunoglobulina G/inmunología , Inmunoglobulina G/uso terapéutico , Inflamación/complicaciones , Inflamación/inmunología , Inflamación/prevención & control , Interleucina-6/sangre , Interleucina-6/inmunología , Masculino , Serina Proteasas Asociadas a la Proteína de Unión a la Manosa/antagonistas & inhibidores , Serina Proteasas Asociadas a la Proteína de Unión a la Manosa/inmunología , Serina Proteasas Asociadas a la Proteína de Unión a la Manosa/metabolismo , Persona de Mediana Edad , Evaluación de Resultado en la Atención de Salud/métodos , Evaluación de Resultado en la Atención de Salud/estadística & datos numéricos , Estudios Retrospectivos , SARS-CoV-2/fisiología , Microangiopatías Trombóticas/complicaciones , Microangiopatías Trombóticas/inmunologíaRESUMEN
BACKGROUND: IgA nephropathy (IgAN) and Henoch-Schönlein purpura are common glomerular disorders in children sharing the same histopathologic pattern of IgA deposits within the mesangium, even if their physiopathology may be different. Repeated exposure to pathogens induces the production of abnormal IgA1. The immune complex deposition in the renal mesangium in IgAN or potentially in small vessels in Henoch-Schönlein purpura induces complement activation via the alternative and lectin pathways. Recent studies suggest that levels of membrane attack complex (MAC) in the urine might be a useful indicator of renal injury. Because of the emerging availability of therapies that selectively block complement activation, the aim of the present study is to investigate whether MAC immunostaining might be a useful marker of IgA-mediated renal injury. METHODS: We conducted immunohistochemistry analysis of the MAC on renal biopsies from 67 pediatric patients with IgAN and Henoch-Schönlein purpura. We classified their renal biopsies according to the Oxford classification, retrieved symptoms, biological parameters, treatment, and follow-up. RESULTS: We found MAC expression was significantly related to impaired renal function and patients whose clinical course required therapy. MAC deposits tend to be more abundant in patients with decreased glomerular filtration rate (p = 0.02), patients with proteinuria > 0.750 g/day/1.73 m2, and with nephrotic syndrome. No correlation with histological alterations was observed. CONCLUSIONS: We conclude that MAC deposition could be a useful additional indicator of renal injury in patients with IgAN and Henoch-Schönlein purpura, independent of other indicators.
Asunto(s)
Complejo de Ataque a Membrana del Sistema Complemento/análisis , Mesangio Glomerular/patología , Glomerulonefritis por IGA/diagnóstico , Vasculitis por IgA/diagnóstico , Inmunosupresores/uso terapéutico , Adolescente , Biomarcadores/análisis , Biopsia , Niño , Preescolar , Complejo de Ataque a Membrana del Sistema Complemento/inmunología , Vía Alternativa del Complemento/efectos de los fármacos , Vía Alternativa del Complemento/inmunología , Lectina de Unión a Manosa de la Vía del Complemento/efectos de los fármacos , Lectina de Unión a Manosa de la Vía del Complemento/inmunología , Estudios de Factibilidad , Femenino , Estudios de Seguimiento , Mesangio Glomerular/inmunología , Glomerulonefritis por IGA/tratamiento farmacológico , Glomerulonefritis por IGA/inmunología , Glomerulonefritis por IGA/patología , Humanos , Vasculitis por IgA/tratamiento farmacológico , Vasculitis por IgA/inmunología , Vasculitis por IgA/patología , Inmunoglobulina A/inmunología , Inmunosupresores/farmacología , Masculino , Pronóstico , Estudios Retrospectivos , Resultado del TratamientoRESUMEN
Mannose-binding lectin (MBL), an initiator of the lectin pathway, is detrimental in ischemic stroke. MBL deposition on the ischemic endothelium indicates the beginning of its actions, but downstream mechanisms are not clear yet.We investigated MBL interactions with the ischemic endothelium by exposing human brain microvascular endothelial cells (hBMECs) to protocols of ischemia. Cells were exposed to hypoxia or oxygen-glucose deprivation (OGD), and re-oxygenated with human serum (HS) or recombinant MBL (rhMBL). Hypoxic hBMECs re-oxygenated with HS showed increased complement system activation (C3c deposition, +59%) and MBL deposition (+93%) than normoxic cells. Super-resolution microscopy showed MBL internalization in hypoxic cells and altered cytoskeletal organization, indicating a potential MBL action on the endothelial structure. To isolate MBL effect, hBMECs were re-oxygenated with rhMBL after hypoxia/OGD. In both conditions, MBL reduced viability (hypoxia: -25%, OGD: -34%) compared to conditions without MBL, showing a direct toxic effect. Ischemic cells also showed greater MBL deposition (hypoxia: +143%, OGD: +126%) than normoxic cells. These results were confirmed with primary hBMECs exposed to OGD (increased MBL-induced cell death: +226%, and MBL deposition: +104%). The present findings demonstrate that MBL can exert a direct deleterious effect on ischemic brain endothelial cells in vitro, independently from complement activation.
Asunto(s)
Isquemia Encefálica/metabolismo , Células Endoteliales/efectos de los fármacos , Endotelio Vascular/efectos de los fármacos , Lectina de Unión a Manosa/metabolismo , Isquemia Encefálica/patología , Hipoxia de la Célula/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Lectina de Unión a Manosa de la Vía del Complemento/efectos de los fármacos , Células Endoteliales/metabolismo , Células Endoteliales/patología , Endotelio Vascular/metabolismo , Endotelio Vascular/patología , Glucosa/metabolismo , Humanos , Lectina de Unión a Manosa/genética , Lectina de Unión a Manosa/farmacología , Oxígeno/metabolismo , Cultivo Primario de Células , Suero/metabolismoRESUMEN
The complement system is a powerful mechanism of the innate immune defense system. Dysregulation may contribute to several diseases. Heparin is a known regulator of the complement system, but its application is limited due to its anticoagulative activity. A promising alternative is the synthetic analogue dendritic polyglycerol sulfate (dPGS). Although dPGS-mediated inhibition of the classical and alternative pathway has been roughly described previously, here we analyzed the effects of dPGS regarding the three pathways at different levels of the proteolytic cascades for the first time. Regarding the final outcome (membrane attack complex formation), IC50 values for dPGS varied between the alternative (900 nM), the classical (300 nM), and the lectin pathway (60 nM). In a backward approach, processing of proteins C5 and C3 via the respective convertase was analyzed by ELISA to narrow down dPGS targets. A dose-dependent reduction of C5a and C3a levels was detected. Further, the analysis via surface plasmon resonance revealed novel dPGS binding proteins; the pro-inflammatory anaphylatoxins C3a and C5a and the classical pathway activator C1q showed down to nanomolar binding affinities. The fully synthetic multivalent polymer dPGS seems to be a promising candidate for the further development to counteract excessive complement activation in disease.
Asunto(s)
Complemento C3/antagonistas & inhibidores , Complemento C5/antagonistas & inhibidores , Glicerol/farmacología , Polímeros/farmacología , Vía Alternativa del Complemento/efectos de los fármacos , Vía Clásica del Complemento/efectos de los fármacos , Lectina de Unión a Manosa de la Vía del Complemento/efectos de los fármacos , Glicerol/química , Humanos , Polímeros/química , Proteolisis/efectos de los fármacosRESUMEN
The complement system has moved into the focus of drug development efforts in the last decade, since its inappropriate or uncontrolled activation has been recognized in many diseases. Some of them are primarily complement-mediated rare diseases, such as paroxysmal nocturnal hemoglobinuria, C3 glomerulonephritis, and atypical hemolytic uremic syndrome. Complement also plays a role in various multifactorial diseases that affect millions of people worldwide, such as ischemia reperfusion injury (myocardial infarction, stroke), age-related macular degeneration, and several neurodegenerative disorders. In this review, we summarize the potential advantages of targeting various complement proteins with special emphasis on the components of the lectin (LP) and the alternative pathways (AP). The serine proteases (MASP-1/2/3, factor D, factor B), which are responsible for the activation of the cascade, are straightforward targets of inhibition, but the pattern recognition molecules (mannose-binding lectin, other collectins, and ficolins), the regulatory components (factor H, factor I, properdin), and C3 are also subjects of drug development. Recent discoveries about cross-talks between the LP and AP offer new approaches for clinical intervention. Mannan-binding lectin-associated serine proteases (MASPs) are not just responsible for LP activation, but they are also indispensable for efficient AP activation. Activated MASP-3 has recently been shown to be the enzyme that continuously supplies factor D (FD) for the AP by cleaving pro-factor D (pro-FD). In this aspect, MASP-3 emerges as a novel feasible target for the regulation of AP activity. MASP-1 was shown to be required for AP activity on various surfaces, first of all on LPS of Gram-negative bacteria.
Asunto(s)
Vía Alternativa del Complemento/efectos de los fármacos , Lectina de Unión a Manosa de la Vía del Complemento/efectos de los fármacos , Proteínas del Sistema Complemento/inmunología , Enfermedades del Sistema Inmune/tratamiento farmacológico , Enfermedades del Sistema Inmune/inmunología , Animales , Biomarcadores , Proteínas del Sistema Complemento/metabolismo , Descubrimiento de Drogas , Humanos , Enfermedades del Sistema Inmune/metabolismo , Terapia Molecular Dirigida , Transducción de Señal , Relación Estructura-ActividadRESUMEN
Acute myocardial infarction (AMI) remains a leading cause of morbidity and mortality in modern medicine. Early reperfusion accomplished by primary percutaneous coronary intervention is pivotal for reducing myocardial damage in ST elevation AMI. However, restoration of coronary blood flow may paradoxically trigger cardiomyocyte death secondary to a reperfusion-induced inflammatory process, which may account for a significant proportion of the final infarct size. Unfortunately, recent human trials targeting myocardial ischemia/reperfusion (I/R) injury have yielded disappointing results. In experimental models of myocardial I/R injury, the complement system, and in particular the lectin pathway, have been identified as major contributors. In line with this, C1 esterase inhibitor (C1INH), the natural inhibitor of the lectin pathway, was shown to significantly ameliorate myocardial I/R injury. However, the hypothesis of a considerable augmentation of myocardial I/R injury by activation of the lectin pathway has not yet been confirmed in humans, which questions the efficacy of a therapeutic strategy solely aimed at the inhibition of the lectin pathway after human AMI. Thus, as C1INH is a multiple-action inhibitor targeting several pathways and mediators simultaneously in addition to the lectin pathway, such as the contact and coagulation system and tissue leukocyte infiltration, this may be considered as being advantageous over exclusive inhibition of the lectin pathway. In this review, we summarize current concepts and evidence addressing the role of the lectin pathway as a potent mediator/modulator of myocardial I/R injury in animal models and in patients. In addition, we focus on the evidence and the potential advantages of using the natural inhibitor of the lectin pathway, C1INH, as a future therapeutic approach in AMI given its ability to interfere with several plasmatic cascades. Ameliorating myocardial I/R injury by targeting the complement system and other plasmatic cascades remains a valid option for future therapeutic interventions.
Asunto(s)
Lectina de Unión a Manosa de la Vía del Complemento/inmunología , Daño por Reperfusión Miocárdica/etiología , Daño por Reperfusión Miocárdica/metabolismo , Animales , Biomarcadores , Proteína Inhibidora del Complemento C1/metabolismo , Lectina de Unión a Manosa de la Vía del Complemento/efectos de los fármacos , Humanos , Terapia Molecular Dirigida , Daño por Reperfusión Miocárdica/tratamiento farmacológico , Daño por Reperfusión Miocárdica/patología , Resultado del TratamientoRESUMEN
Oncological treatment has been associated with an increased risk of infection, most often related to therapy-induced pancytopenia. However, limited research has been conducted on the effect of oncological therapy on the complement system, being part of the non-cellular innate immune system. This became the rationale for an observational clinical study (C2012) in which we have investigated the prevalence of transient complement defects. Once we had observed such defects, a correlation of the complement defects to specific clinical parameters or to specific therapeutic regimens was investigated. A prominent defect observed in C2012 was the inhibition of the lectin pathway (LP) of complement activation during the treatment of acute lymphoblastic leukemia (ALL), which we could directly associate to the use of asparaginase (ASNase). Ex-vivo experiments confirmed a direct dose-dependent inhibitory effect of ASNase on the LP functionality.
Asunto(s)
Asparaginasa/farmacología , Lectina de Unión a Manosa de la Vía del Complemento/efectos de los fármacos , Polietilenglicoles/farmacología , Asparaginasa/administración & dosificación , Asparaginasa/uso terapéutico , Niño , Depresión Química , Relación Dosis-Respuesta a Droga , Humanos , Lectina de Unión a Manosa/sangre , Serina Proteasas Asociadas a la Proteína de Unión a la Manosa/antagonistas & inhibidores , Polietilenglicoles/administración & dosificación , Polietilenglicoles/uso terapéutico , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Unión Proteica/efectos de los fármacosRESUMEN
Complement is crucial to the immune response, but dysregulation of the system causes inflammatory disease. Complement is activated by three pathways: classical, lectin, and alternative. The classical and lectin pathways are initiated by the C1r/C1s (classical) and MASP-1/MASP-2 (lectin) proteases. Given the role of complement in disease, there is a requirement for inhibitors to control the initiating proteases. In this article, we show that a novel inhibitor, gigastasin, from the giant Amazon leech, potently inhibits C1s and MASP-2, whereas it is also a good inhibitor of MASP-1. Gigastasin is a poor inhibitor of C1r. The inhibitor blocks the active sites of C1s and MASP-2, as well as the anion-binding exosites of the enzymes via sulfotyrosine residues. Complement deposition assays revealed that gigastasin is an effective inhibitor of complement activation in vivo, especially for activation via the lectin pathway. These data suggest that the cumulative effects of inhibiting both MASP-2 and MASP-1 have a greater effect on the lectin pathway than the more potent inhibition of only C1s of the classical pathway.
Asunto(s)
Activación de Complemento/efectos de los fármacos , Complemento C1/antagonistas & inhibidores , Inactivadores del Complemento/química , Vía Clásica del Complemento/efectos de los fármacos , Lectina de Unión a Manosa de la Vía del Complemento/efectos de los fármacos , Sanguijuelas/química , Serina Proteasas Asociadas a la Proteína de Unión a la Manosa/antagonistas & inhibidores , Péptidos/química , Inhibidores de Serina Proteinasa/química , Animales , Dominio Catalítico/efectos de los fármacos , Células Cultivadas , Inactivadores del Complemento/farmacología , Endotelio Vascular/efectos de los fármacos , Humanos , Péptidos/farmacología , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacología , Inhibidores de Serina Proteinasa/farmacologíaRESUMEN
Physiochemical stress induces tissue injury as a result of the detection of abnormal molecular patterns by sensory molecules of the innate immune system. Here, we have described how the recently discovered C-type lectin collectin-11 (CL-11, also known as CL-K1 and encoded by COLEC11) recognizes an abnormal pattern of L-fucose on postischemic renal tubule cells and activates a destructive inflammatory response. We found that intrarenal expression of CL-11 rapidly increases in the postischemic period and colocalizes with complement deposited along the basolateral surface of the proximal renal tubule in association with L-fucose, the potential binding ligand for CL-11. Mice with either generalized or kidney-specific deficiency of CL-11 were strongly protected against loss of renal function and tubule injury due to reduced complement deposition. Ex vivo renal tubule cells showed a marked capacity for CL-11 binding that was induced by cell stress under hypoxic or hypothermic conditions and prevented by specific removal of L-fucose. Further analysis revealed that cell-bound CL-11 required the lectin complement pathway-associated protease MASP-2 to trigger complement deposition. Given these results, we conclude that lectin complement pathway activation triggered by ligand-CL-11 interaction in postischemic tissue is a potent source of acute kidney injury and is amenable to sugar-specific blockade.
Asunto(s)
Lesión Renal Aguda/metabolismo , Colectinas/metabolismo , Lectina de Unión a Manosa de la Vía del Complemento/efectos de los fármacos , Proteínas del Sistema Complemento/metabolismo , Fucosa/toxicidad , Túbulos Renales Proximales/lesiones , Túbulos Renales Proximales/metabolismo , Lesión Renal Aguda/inducido químicamente , Lesión Renal Aguda/genética , Lesión Renal Aguda/patología , Animales , Colectinas/genética , Lectina de Unión a Manosa de la Vía del Complemento/genética , Proteínas del Sistema Complemento/genética , Túbulos Renales Proximales/patología , Serina Proteasas Asociadas a la Proteína de Unión a la Manosa/genética , Serina Proteasas Asociadas a la Proteína de Unión a la Manosa/metabolismo , Ratones , Ratones NoqueadosRESUMEN
Complement activation is of major importance in numerous pathological conditions. Therefore, targeted complement inhibition is a promising therapeutic strategy. C1-esterase inhibitor (C1-INH) controls activation of the classical pathway (CP) and the lectin pathway (LP). However, conflicting data exist on inhibition of the alternative pathway (AP) by C1-INH. The inhibitory capacity of C1-INH for the CP is potentiated by heparin and other glycosaminoglycans, but no data exist for the LP and AP. The current study investigates the effects of C1-INH in the presence or absence of different clinically used heparinoids on the CP, LP and AP. Furthermore, the combined effects of heparinoids and C1-INH on coagulation were investigated. C1-INH, heparinoids or combinations were analysed in a dose-dependent fashion in the presence of pooled serum. Functional complement activities were measured simultaneously using the Wielisa(®) -kit. The activated partial thrombin time was determined using an automated coagulation analyser. The results showed that all three complement pathways were inhibited significantly by C1-INH or heparinoids. Next to their individual effects on complement activation, heparinoids also enhanced the inhibitory capacity of C1-INH significantly on the CP and LP. For the AP, significant potentiation of C1-INH by heparinoids was found; however, this was restricted to certain concentration ranges. At low concentrations the effect on blood coagulation by combining heparinoids with C1-INH was minimal. In conclusion, our study shows significant potentiating effects of heparinoids on the inhibition of all complement pathways by C1-INH. Therefore, their combined use is a promising and a potentially cost-effective treatment option for complement-mediated diseases.
Asunto(s)
Activación de Complemento/efectos de los fármacos , Proteína Inhibidora del Complemento C1/farmacología , Heparinoides/farmacología , Coagulación Sanguínea/efectos de los fármacos , Vía Alternativa del Complemento/efectos de los fármacos , Vía Clásica del Complemento/efectos de los fármacos , Lectina de Unión a Manosa de la Vía del Complemento/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Combinación de Medicamentos , Sinergismo Farmacológico , Humanos , Tiempo de Tromboplastina ParcialRESUMEN
The group B Streptococcus (GBS) is a leading cause of neonatal invasive disease. GBS bacteria are surrounded by a thick capsular polysaccharide that is a potent inhibitor of complement deposition via the alternative pathway. Several of its surface molecules can however activate the classical and lectin complement pathways, rendering this species still vulnerable to phagocytic killing. In this study we have identified a novel secreted protein named complement interfering protein (CIP) that downregulates complement activation via the classical and lectin pathways, but not the alternative pathway. The CIP protein showed high affinity toward C4b and inhibited its interaction with C2, presumably preventing the formation of the C4bC2a convertase. Addition of recombinant CIP to GBS cip-negative bacteria resulted in decreased deposition of C3b on their surface and in diminished phagocytic killing in a whole-blood assay. Our data reveal a novel strategy exploited by GBS to counteract innate immunity and could be valuable for the development of anti-infective agents against this important pathogen.
Asunto(s)
Proteínas Bacterianas/inmunología , Complemento C4b/inmunología , Vía Clásica del Complemento/inmunología , Lectina de Unión a Manosa de la Vía del Complemento/inmunología , Evasión Inmune/inmunología , Streptococcus agalactiae/inmunología , Adulto , Secuencia de Aminoácidos , Activación de Complemento/inmunología , Complemento C3b/biosíntesis , Complemento C3b/inmunología , Vía Alternativa del Complemento/inmunología , Vía Clásica del Complemento/efectos de los fármacos , Lectina de Unión a Manosa de la Vía del Complemento/efectos de los fármacos , Humanos , Inmunidad Innata , Datos de Secuencia Molecular , Fagocitosis/inmunología , Unión Proteica/inmunología , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacología , Streptococcus agalactiae/genética , Streptococcus agalactiae/metabolismoRESUMEN
The abortion-prone mating combination CBA/J × DBA/2 has been recognized as a model of preeclampsia, and complement activation has been implicated in the high rate of pregnancy loss observed in CBA/J mice. We have analyzed the implantation sites collected from DBA/2-mated CBA/J mice for the deposition of the complement recognition molecules using CBA/J mated with BALB/c mice as a control group. MBL-A was observed in the implantation sites of CBA/J × DBA/2 combination in the absence of MBL-C and was undetectable in BALB/c-mated CBA/J mice. Conversely, C1q was present in both mating combinations. Searching for other complement components localized at the implantation sites of CBA/J × DBA/2, we found C4 and C3, but we failed to reveal C1r. These data suggest that complement is activated through the lectin pathway and proceeds to completion of the activation sequence as revealed by C9 deposition. MBL-A was detected as early as 3.5 d of pregnancy, and MBL-A deficiency prevented pregnancy loss in the abortion-prone mating combination. The contribution of the terminal complex to miscarriage was supported by the finding that pregnancy failure was largely inhibited by the administration of neutralizing Ab to C5. Treatment of DBA/2-mated CBA/J mice with Polyman2 that binds to MBL-A with high affinity proved to be highly effective in controlling the activation of the lectin pathway and in preventing fetal loss.
Asunto(s)
Lectina de Unión a Manosa de la Vía del Complemento , Preeclampsia/tratamiento farmacológico , Animales , Anticuerpos Bloqueadores/administración & dosificación , Complemento C5/inmunología , Complemento C5/metabolismo , Lectina de Unión a Manosa de la Vía del Complemento/efectos de los fármacos , Modelos Animales de Enfermedad , Implantación del Embrión/efectos de los fármacos , Femenino , Humanos , Masculino , Lectina de Unión a Manosa/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos CBA , Ratones Endogámicos DBA , Preeclampsia/inmunología , EmbarazoRESUMEN
Off-target binding connotes the binding of a small molecule of therapeutic significance to a protein target in addition to the primary target for which it was proposed. Progressively such off-targeting is emerging to be regular practice to reveal side effects. Chymase is an enzyme of hydrolase class that catalyzes hydrolysis of peptide bonds. A link between heart failure and chymase is ascribed, and a chymase inhibitor is in clinical phase II for treatment of heart failure. However, the underlying mechanisms of the off-target effects of human chymase inhibitors are still unclear. Here, we develop a robust computational strategy that is applicable to any enzyme system and that allows the prediction of drug effects on biological processes. Putative off-targets for chymase inhibitors were identified through various structural and functional similarity analyses along with molecular docking studies. Finally, literature survey was performed to incorporate these off-targets into biological pathways and to establish links between pathways and particular adverse effects. Off-targets of chymase inhibitors are linked to various biological pathways such as classical and lectin pathways of complement system, intrinsic and extrinsic pathways of coagulation cascade, and fibrinolytic system. Tissue kallikreins, granzyme M, neutrophil elastase, and mesotrypsin are also identified as off-targets. These off-targets and their associated pathways are elucidated for the effects of inflammation, cancer, hemorrhage, thrombosis, and central nervous system diseases (Alzheimer's disease). Prospectively, our approach is helpful not only to better understand the mechanisms of chymase inhibitors but also for drug repurposing exercises to find novel uses for these inhibitors.
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Quimasas/antagonistas & inhibidores , Inhibidores Enzimáticos/química , Simulación del Acoplamiento Molecular , Bibliotecas de Moléculas Pequeñas/química , Biología de Sistemas/métodos , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/enzimología , Enfermedad de Alzheimer/patología , Coagulación Sanguínea/efectos de los fármacos , Enfermedades Cardiovasculares/tratamiento farmacológico , Enfermedades Cardiovasculares/enzimología , Enfermedades Cardiovasculares/patología , Quimasas/química , Quimasas/metabolismo , Lectina de Unión a Manosa de la Vía del Complemento/efectos de los fármacos , Diseño de Fármacos , Inhibidores Enzimáticos/farmacología , Fibrinólisis/efectos de los fármacos , Granzimas/antagonistas & inhibidores , Granzimas/química , Granzimas/metabolismo , Humanos , Elastasa de Leucocito/antagonistas & inhibidores , Elastasa de Leucocito/química , Elastasa de Leucocito/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacología , Relación Estructura-Actividad , Calicreínas de Tejido/antagonistas & inhibidores , Calicreínas de Tejido/química , Calicreínas de Tejido/metabolismo , Tripsina/química , Tripsina/metabolismo , Interfaz Usuario-ComputadorRESUMEN
BACKGROUND: The complement system is a key component of innate immunity implicated in the neutralization and clearance of invading pathogens. Dextran coated superparamagnetic iron oxide (SPIO) nanoparticle is a promising magnetic resonance imaging (MRI) contrast agent. However, dextran SPIO has been associated with significant number of complement-related side effects in patients and some agents have been discontinued from clinical use (e.g., Feridex™). In order to improve the safety of these materials, the mechanisms of complement activation by dextran-coated SPIO and the differences between mice and humans need to be fully understood. METHODS: 20 kDa dextran coated SPIO nanoworms (SPIO NW) were synthesized using Molday precipitation procedure. In vitro measurements of C3 deposition on SPIO NW using sera genetically deficient for various components of the classical pathway (CP), lectin pathway (LP) or alternative pathway (AP) components were used to study mechanisms of mouse complement activation. In vitro measurements of fluid phase markers of complement activation C4d and Bb and the terminal pathway marker SC5b-C9 in normal and genetically deficient sera were used to study the mechanisms of human complement activation. Mouse data were analyzed by non-paired t-test, human data were analyzed by ANOVA followed by multiple comparisons with Student-Newman-Keuls test. RESULTS: In mouse sera, SPIO NW triggered the complement activation via the LP, whereas the AP contributes via the amplification loop. No involvement of the CP was observed. In human sera the LP together with the direct enhancement of the AP turnover was responsible for the complement activation. In two samples out of six healthy donors there was also a binding of anti-dextran antibodies and C1q, suggesting activation via the CP, but that did not affect the total level of C3 deposition on the particles. CONCLUSIONS: There were important differences and similarities in the complement activation by SPIO NW in mouse versus human sera. Understanding the mechanisms of immune recognition of nanoparticles in mouse and human systems has important preclinical and clinical implications and could help design more efficient and safe nano-formulations.
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Activación de Complemento/efectos de los fármacos , Medios de Contraste/farmacología , Dextranos/farmacología , Adulto , Animales , Biomarcadores/sangre , Vía Alternativa del Complemento/efectos de los fármacos , Vía Clásica del Complemento/efectos de los fármacos , Lectina de Unión a Manosa de la Vía del Complemento/efectos de los fármacos , Proteínas del Sistema Complemento/genética , Proteínas del Sistema Complemento/metabolismo , Humanos , Nanopartículas de Magnetita , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Especificidad de la Especie , Propiedades de SuperficieRESUMEN
Paroxysmal nocturnal hemoglobinuria (PNH) is a rare, clonal, hematopoietic stem cell disorder that manifests with a complement-mediated hemolytic anemia, bone marrow failure, and a propensity for thrombosis. These patients experience both intra- and extravascular hemolysis in the context of underlying complement activation. Currently eculizumab effectively blocks the intravascular hemolysis PNH. There remains an unmet clinical need for a complement inhibitor with activity early in the complement cascade to block complement at the classical and alternative pathways. C1 esterase inhibitor (C1INH) is an endogenous human plasma protein that has broad inhibitory activity in the complement pathway through inhibition of the classical pathway by binding C1r and C1s and inhibits the mannose-binding lectin-associated serine proteases in the lectin pathway. In this study, we show that commercially available plasma derived C1INH prevents lysis induced by the alternative complement pathway of PNH erythrocytes in human serum. Importantly, C1INH was able to block the accumulation of C3 degradation products on CD55 deficient erythrocytes from PNH patient on eculizumab therapy. This could suggest a role for inhibition of earlier phases of the complement cascade than that currently inhibited by eculizumab for incomplete or nonresponders to that therapy.
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Proteína Inhibidora del Complemento C1/farmacología , Vía Clásica del Complemento/efectos de los fármacos , Lectina de Unión a Manosa de la Vía del Complemento/efectos de los fármacos , Hemoglobinuria Paroxística/tratamiento farmacológico , Adulto , Anticuerpos Monoclonales Humanizados/farmacología , Anticuerpos Monoclonales Humanizados/uso terapéutico , Antígenos CD55/sangre , Antígenos CD59/sangre , Complemento C3/metabolismo , Complemento C5/antagonistas & inhibidores , Vía Alternativa del Complemento/fisiología , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Resistencia a Medicamentos , Membrana Eritrocítica/efectos de los fármacos , Membrana Eritrocítica/inmunología , Membrana Eritrocítica/metabolismo , Femenino , Hemoglobinuria Paroxística/sangre , Hemoglobinuria Paroxística/inmunología , Humanos , Masculino , Persona de Mediana Edad , Adulto JovenRESUMEN
Scabies is an infectious skin disease caused by the mite Sarcoptes scabiei and has been classified as one of the six most prevalent epidermal parasitic skin diseases infecting populations living in poverty by the World Health Organisation. The role of the complement system, a pivotal component of human innate immunity, as an important defence against invading pathogens has been well documented and many parasites have an arsenal of anti-complement defences. We previously reported on a family of scabies mite proteolytically inactive serine protease paralogues (SMIPP-Ss) thought to be implicated in host defence evasion. We have since shown that two family members, SMIPP-S D1 and I1 have the ability to bind the human complement components C1q, mannose binding lectin (MBL) and properdin and are capable of inhibiting all three human complement pathways. This investigation focused on inhibition of the lectin pathway of complement activation as it is likely to be the primary pathway affecting scabies mites. Activation of the lectin pathway relies on the activation of MBL, and as SMIPP-S D1 and I1 have previously been shown to bind MBL, the nature of this interaction was examined using binding and mutagenesis studies. SMIPP-S D1 bound MBL in complex with MBL-associated serine proteases (MASPs) and released the MASP-2 enzyme from the complex. SMIPP-S I1 was also able to bind MBL in complex with MASPs, but MASP-1 and MASP-2 remained in the complex. Despite these differences in mechanism, both molecules inhibited activation of complement components downstream of MBL. Mutagenesis studies revealed that both SMIPP-Ss used an alternative site of the molecule from the residual active site region to inhibit the lectin pathway. We propose that SMIPP-Ss are potent lectin pathway inhibitors and that this mechanism represents an important tool in the immune evasion repertoire of the parasitic mite and a potential target for therapeutics.
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Lectina de Unión a Manosa de la Vía del Complemento/efectos de los fármacos , Sarcoptes scabiei/enzimología , Serina Proteasas/metabolismo , Serina Proteasas/farmacología , Secuencia de Aminoácidos , Animales , Complemento C1q/antagonistas & inhibidores , Complemento C1q/metabolismo , Interacciones Huésped-Patógeno , Humanos , Lectina de Unión a Manosa/antagonistas & inhibidores , Lectina de Unión a Manosa/metabolismo , Serina Proteasas Asociadas a la Proteína de Unión a la Manosa/metabolismo , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación , Escabiosis/metabolismo , Alineación de Secuencia , Serina Proteasas/química , Serina Proteasas/genéticaRESUMEN
OBJECTIVES: To assess the involvement of ficolin-3, the main initiator of the lectin complement pathway (LCP), in subarachnoid hemorrhage (SAH) pathology and outcome. METHODS: In this preliminary exploratory study, plasma concentration of ficolin-3 and of ficolin-3-mediated functional LCP activity was measured, along with that of other LCP initiators (mannose-binding lectin, ficolin-2, and ficolin-1), C3 activation products, and soluble C5b-9 terminal complex, in a prospective cohort of 39 patients with SAH and 20 healthy controls. The following parameters were recorded: SAH severity, assessed using the World Federation of Neurosurgical Societies grading scale; vasospasm, defined as neuro-worsening with angiographic confirmation of vessel narrowing; cerebral ischemia, defined as hypodense lesion on CT scan performed before discharge; and 6-month outcome, assessed using the Glasgow Outcome Scale. RESULTS: In patients, no changes were detected for ficolin-3 compared with controls. Notably, however, ficolin-3-mediated functional LCP activity was reduced. Low levels of plasma ficolin-3 and ficolin-3-mediated functional LCP activity were related to SAH severity, vasospasm, and cerebral ischemia. Moreover, ficolin-3 functional LCP activity was decreased in patients with unfavorable outcome. CONCLUSION: Our data provide evidence that LCP is activated after SAH and that the actual plasma concentrations of ficolin-3 reflect the severity of brain injury as evaluated by clinical and structural parameters. These results support the idea that ficolin-3-mediated functional LCP activity may be targeted to control injury progression in SAH.
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Lectina de Unión a Manosa de la Vía del Complemento/fisiología , Glicoproteínas/fisiología , Lectinas/fisiología , Hemorragia Subaracnoidea/sangre , Anciano , Isquemia Encefálica/etiología , Isquemia Encefálica/fisiopatología , Isquemia Encefálica/cirugía , Estudios de Cohortes , Lectina de Unión a Manosa de la Vía del Complemento/efectos de los fármacos , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Lectinas/farmacología , Masculino , Persona de Mediana Edad , Procedimientos Neuroquirúrgicos , Estudios Prospectivos , Hemorragia Subaracnoidea/patología , Hemorragia Subaracnoidea/cirugía , Tomografía Computarizada por Rayos X , Resultado del Tratamiento , Vasoespasmo Intracraneal/etiología , Vasoespasmo Intracraneal/fisiopatología , Vasoespasmo Intracraneal/cirugía , FicolinasRESUMEN
Activation of complement may cause severe tissue damage in antibody-mediated allograft rejection and other antibody-mediated clinical conditions; therefore, novel potent complement inhibitors are needed. Previously, we described binding of the inhibitory receptor LAIR-1 and its soluble family member LAIR-2 to collagen. Here, we investigated binding of LAIR-1 and LAIR-2 to the complement proteins C1q and MBL, which both have collagen-like domains, and evaluated the effect of this binding on complement function. We demonstrate specific binding of recombinant LAIR proteins to both C1q and MBL. Surface plasmon resonance experiments showed that LAIR-2-Fc protein bound C1q and MBL with the highest affinity compared to LAIR-2-HIS. We, therefore, hypothesized that LAIR-2-Fc is a potent complement inhibitor. Indeed, LAIR-2-Fc inhibited C4 fixation to IgG or mannan, reduced activation of C4 by aggregated IgG in plasma and inhibited iC3b deposition on cells. Finally, LAIR-2-Fc inhibited complement-mediated lysis of cells sensitized with anti-HLA antibodies in an ex vivo model for antibody-mediated transplant rejection. Thus, LAIR-2-Fc is an effective novel complement inhibitor for the treatment and prevention of antibody-mediated allograft rejection and antibody-mediated clinical conditions.
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Rechazo de Injerto/prevención & control , Enfermedades del Complejo Inmune/terapia , Inmunoterapia , Receptores Inmunológicos/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Citotoxicidad Celular Dependiente de Anticuerpos/efectos de los fármacos , Complemento C1q/metabolismo , Lectina de Unión a Manosa de la Vía del Complemento/efectos de los fármacos , Rechazo de Injerto/inmunología , Antígenos HLA/inmunología , Humanos , Enfermedades del Complejo Inmune/inmunología , Regiones Constantes de Inmunoglobulina/genética , Isoanticuerpos/metabolismo , Células K562 , Unión Proteica/efectos de los fármacos , Receptores Inmunológicos/administración & dosificación , Receptores Inmunológicos/genética , Proteínas Recombinantes de Fusión/administración & dosificación , Proteínas Recombinantes de Fusión/genéticaRESUMEN
Previous experiments from our laboratories have identified peptides derived from the human astrovirus coat protein (CP) that bind C1q and mannose binding lectin (MBL) inhibiting activation of the classical and lectin pathways of complement, respectively. The purpose of this study was to evaluate the function of these coat protein peptides (CPPs) in an in vitro model of complement-mediated disease (ABO incompatibility), preliminarily assess their in vivo complement suppression profile and develop more highly potent derivatives of these molecules. E23A, a 30 amino acid CPP derivative previously demonstrated to inhibit classical pathway activation was able to dose-dependently inhibit lysis of AB erythrocytes treated with mismatched human O serum. Additionally, when injected into rats, E23A inhibited the animals' serum from lysing antibody-sensitized erythrocytes, providing preliminary in vivo functional evidence that this CPP can cross the species barrier to inhibit serum complement activity in rodents. A rational drug design approach was implemented to identify more potent CPP derivatives, resulting in the identification and characterization of a 15 residue peptide (polar assortant (PA)), which demonstrated both superior inhibition of classical complement pathway activation and robust binding to C1q collagen-like tails. PA also inhibited ABO incompatibility in vitro and demonstrated in vivo complement suppression up to 24h post-injection. CPP's ability to inhibit ABO incompatibility in vitro, proof of concept in vivo inhibitory activity in rats and the development of the highly potent PA derivative set the stage for preclinical testing of this molecule in small animal models of complement-mediated disease.