RESUMEN
OBJECTIVE: Innate immune response and particularly terminal complement complex (TCC) deposition are thought to be involved in the pathogenesis of posttraumatic osteoarthritis. However, the possible role of TCC in regulated cell death as well as chondrocyte hypertrophy and senescence has not been unraveled so far and was first addressed using an ex vivo human cartilage trauma-model. DESIGN: Cartilage explants were subjected to blunt impact (0.59 J) and exposed to human serum (HS) and cartilage homogenate (HG) with or without different potential therapeutics: RIPK1-inhibitor Necrostatin-1 (Nec), caspase-inhibitor zVAD, antioxidant N-acetyl cysteine (NAC) and TCC-inhibitors aurintricarboxylic acid (ATA) and clusterin (CLU). Cell death and hypertrophy/senescence-associated markers were evaluated on mRNA and protein level. RESULTS: Addition of HS resulted in significantly enhanced TCC deposition on chondrocytes and decrease of cell viability after trauma. This effect was potentiated by HG and was associated with expression of RIPK3, MLKL and CASP8. Cytotoxicity of HS could be prevented by heat-inactivation or specific inhibitors, whereby combination of Nec and zVAD as well as ATA exhibited highest cell protection. Moreover, HS+HG exposition enhanced the gene expression of CXCL1, IL-8, RUNX2 and VEGFA as well as secretion of IL-6 after cartilage trauma. CONCLUSIONS: Our findings imply crucial involvement of the complement system and primarily TCC in regulated cell death and phenotypic changes of chondrocytes after cartilage trauma. Inhibition of TCC formation or downstream signaling largely modified serum-induced pathophysiologic effects and might therefore represent a therapeutic target to maintain the survival and chondrogenic character of cartilage cells.
Asunto(s)
Muerte Celular/genética , Condrocitos/metabolismo , Complejo de Ataque a Membrana del Sistema Complemento/genética , Hipertrofia/genética , Osteoartritis/genética , Heridas no Penetrantes/genética , Acetilcisteína/farmacología , Anciano , Anciano de 80 o más Años , Ácido Aurintricarboxílico/farmacología , Cartílago Articular/citología , Muerte Celular/efectos de los fármacos , Senescencia Celular/efectos de los fármacos , Senescencia Celular/genética , Condrocitos/efectos de los fármacos , Condrocitos/patología , Clusterina/farmacología , Complejo de Ataque a Membrana del Sistema Complemento/antagonistas & inhibidores , Complejo de Ataque a Membrana del Sistema Complemento/efectos de los fármacos , Complejo de Ataque a Membrana del Sistema Complemento/metabolismo , Inhibidores Enzimáticos/farmacología , Femenino , Depuradores de Radicales Libres/farmacología , Humanos , Imidazoles/farmacología , Inmunidad Innata/genética , Indoles/farmacología , Masculino , Persona de Mediana Edad , Oligopéptidos/farmacología , Osteoartritis/etiología , Osteoartritis/metabolismo , Osteoartritis/patología , ARN Mensajero/efectos de los fármacos , ARN Mensajero/metabolismo , Heridas no Penetrantes/complicaciones , Heridas no Penetrantes/metabolismoRESUMEN
Age Related Macular Degeneration (AMD) is the first cause of social blindness in people aged over 65 leading to atrophy of retinal pigment epithelial cells (RPE), photoreceptors and choroids, eventually associated with choroidal neovascularization. Accumulation of undigested cellular debris within RPE cells or under the RPE (Drusen), oxidative stress and inflammatory mediators contribute to the RPE cell death. The major risk to develop AMD is the Y402H polymorphism of complement factor H (CFH). CFH interacting with oxidized phospholipids on the RPE membrane modulates the functions of these cells, but the exact role of CFH in RPE cell death and survival remain poorly understood. The aim of this study was to analyze the potential protective mechanism of CFH on RPE cells submitted to oxidative stress. Upon exposure to oxidized lipids 4-HNE (4-hydroxy-2-nonenal) derived from photoreceptors, both the human RPE cell line ARPE-19 and RPE cells derived from human induced pluripotent stem cells were protected from death only in the presence of the full length human recombinant CFH in the culture medium. This protective effect was independent from the membrane attack complex (MAC) formation. CFH maintained RPE cells tight junctions' structure and regulated the caspase dependent apoptosis process. These results demonstrated the CFH anti-oxidative stress functions independently of its capacity to inhibit MAC formation.
Asunto(s)
Factor H de Complemento/farmacología , Complejo de Ataque a Membrana del Sistema Complemento/efectos de los fármacos , Epitelio Pigmentado de la Retina/efectos de los fármacos , Aldehídos/farmacología , Apoptosis/efectos de los fármacos , Western Blotting , Caspasas/metabolismo , Muerte Celular/efectos de los fármacos , Línea Celular , Complejo de Ataque a Membrana del Sistema Complemento/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/metabolismo , Microscopía Electrónica de Transmisión , Estrés Oxidativo/efectos de los fármacos , Reacción en Cadena en Tiempo Real de la Polimerasa , Proteínas Recombinantes , Epitelio Pigmentado de la Retina/metabolismo , Uniones Estrechas/efectos de los fármacosRESUMEN
BACKGROUND: Atypical hemolytic uremic syndrome (aHUS) is a disorder of the microvasculature with hemolytic anemia, thrombocytopenia and acute kidney injury. Nowadays, aHUS is successfully treated with eculizumab, a humanized, chimeric IgG2/4 kappa antibody, which binds human complement C5 and blocks generation of C5a and membrane-attack-complex. CASE PRESENTATION: A 25-year-old woman with end stage renal disease due to relapsing atypical hemolytic uremic syndrome had a relapse of the disease during pregnancy. She was treated with eculizumab. We measured reduced formation of the membrane-attack complex in newborn's umbilical cord vein blood using the sensitive and specific Palarasah-Nielsen-ELISA. CONCLUSIONS: Eculizumab treatment of the mother with end stage renal disease may cause reduced innate immunity which could render newborns more susceptible to infections.
Asunto(s)
Anticuerpos Monoclonales Humanizados/uso terapéutico , Síndrome Hemolítico Urémico Atípico/tratamiento farmacológico , Inactivadores del Complemento/uso terapéutico , Complejo de Ataque a Membrana del Sistema Complemento/efectos de los fármacos , Complicaciones del Embarazo/tratamiento farmacológico , Adulto , Anticuerpos Monoclonales Humanizados/metabolismo , Síndrome Hemolítico Urémico Atípico/inmunología , Complemento C3/metabolismo , Complemento C5a/metabolismo , Complemento C9/metabolismo , Inactivadores del Complemento/metabolismo , Complejo de Ataque a Membrana del Sistema Complemento/metabolismo , Ensayo de Inmunoadsorción Enzimática/métodos , Femenino , Sangre Fetal/inmunología , Humanos , Recién Nacido , Fallo Renal Crónico/tratamiento farmacológico , Embarazo , RecurrenciaRESUMEN
Increasing evidence has demonstrated that complement activation is required for ischemiareperfusion injury (IRI)induced hepatic damage, and cobra venom factor (CVF) can deplete the complement components. The aim of the current study was to investigate the effect and intrinsic mechanism of CVF pretreatment on IRIinduced acute hepatic injury in rats. Acute hepatic injury in rats was induced by bone fracture to simulate trauma, followed by hemorrhage for 90 min, and then the rats were resuscitated for a period of 20 min of reperfusion. The survival times under different CVF treatment doses and schedules for rats with IRI were evaluated. Hepatic tissues and serum samples were analyzed for acute hepatic injury, complement activation, inflammatory mediator release and apoptosis at predetermined times and compared between the IRI group and the CVF pretreatment + IRI groups. Compared to the rats with IRI alone, the survival times were significantly improved among rats with IRI receiving a highdose or lowdose CVF pretreatment (all P<0.01). Upon histological examination, severe hepatic damage was observed in the rats with IRI, accompanied by liver function deterioration, complement and membrane attack complex activation, inflammatory mediator release and hepatic cell apoptosis. CVF pretreatment significantly attenuated the hepatic injury through depletion of anaphylatoxic C5a and membrane attack complex C5b9 activation, and subsequent inhibition of inflammatory mediator release and hepatic cell apoptosis (all P<0.05). The results indicated that CVF pretreatment ameliorates IRIinduced acute hepatic injury. However, further studies are required to determine whether this therapy could be a potential agent for the treatment of IRI injuries in clinical settings.
Asunto(s)
Venenos Elapídicos/administración & dosificación , Inflamación/tratamiento farmacológico , Hepatopatías/tratamiento farmacológico , Daño por Reperfusión/tratamiento farmacológico , Animales , Activación de Complemento/efectos de los fármacos , Complemento C5a/efectos de los fármacos , Complemento C5a/metabolismo , Complejo de Ataque a Membrana del Sistema Complemento/efectos de los fármacos , Complejo de Ataque a Membrana del Sistema Complemento/metabolismo , Venenos Elapídicos/química , Fracturas Óseas/complicaciones , Fracturas Óseas/fisiopatología , Humanos , Inflamación/complicaciones , Inflamación/fisiopatología , Hígado/efectos de los fármacos , Hígado/lesiones , Hígado/fisiopatología , Hepatopatías/complicaciones , Ratas , Daño por Reperfusión/complicaciones , Daño por Reperfusión/fisiopatologíaRESUMEN
Background Severe hypertension can induce thrombotic microangiopathy (TMA) in the renal vasculature, the occurrence of which has been linked to mechanical stress to the endothelium. Complement defects may be the culprit of disease in patients who present with severe renal disease and often progress to ESRD, despite BP control.Methods We studied a well defined cohort of 17 patients with hypertension-associated TMA to define the prevalence of complement defects by a specific ex vivo serum-based microvascular endothelial cell assay.Results Compared with normal human serum and samples from patients with hypertensive arterionephrosclerosis, 14 of 16 (87.5%) serum samples collected at presentation from 16 patients with hypertension-associated TMA induced abnormal C5b9 formation on microvascular endothelial cells. We detected rare variants in complement genes in eight of 17 (47%) patients. ESRD occurred in 14 of 17 (82%) patients, and recurrent TMA after transplant occurred in seven of 11 (64%) donor kidneys. Eculizumab improved the renal function in three patients and prevented TMA recurrence in an allograft recipient.Conclusions These observations point to complement defects as the key causative factor of ESRD and recurrent TMA after transplant in patients presenting with severe hypertension. Complement defects can be identified by measurements of complement activation on microvascular endothelial cells, which should substantially influence treatment and prognosis.
Asunto(s)
Anticuerpos Monoclonales Humanizados/uso terapéutico , Activación de Complemento/efectos de los fármacos , Hipertensión/epidemiología , Enfermedades Renales/epidemiología , Microangiopatías Trombóticas/epidemiología , Adulto , Anciano , Estudios de Casos y Controles , Células Cultivadas , Comorbilidad , Complejo de Ataque a Membrana del Sistema Complemento/efectos de los fármacos , Proteínas del Sistema Complemento/efectos de los fármacos , Progresión de la Enfermedad , Células Endoteliales/citología , Células Endoteliales/efectos de los fármacos , Femenino , Humanos , Hipertensión/diagnóstico , Hipertensión/tratamiento farmacológico , Enfermedades Renales/diagnóstico , Enfermedades Renales/tratamiento farmacológico , Fallo Renal Crónico/epidemiología , Fallo Renal Crónico/fisiopatología , Masculino , Persona de Mediana Edad , Pronóstico , Sistema de Registros , Estudios Retrospectivos , Medición de Riesgo , Índice de Severidad de la Enfermedad , Microangiopatías Trombóticas/diagnóstico , Microangiopatías Trombóticas/tratamiento farmacológicoRESUMEN
Membrane regulatory proteins, such as CD46, CD55, and CD59, prevent excess complement activation and to protect cells from damage. Previous investigations confirmed that shear stress in the physiological range was more favorable for endothelial progenitor cells (EPCs) to repair injured vascular endothelial cells and operates mainly in atheroprotective actions. However, detailed events that contribute to shear stress-induced protection in EPCs, particularly the mechanisms of signal transduction, remain poorly understood. In this study, we observed shear stress-mediated changes in the expression of complement regulatory proteins CD46, CD55, and CD59 on human EPCs and focused on the mechanical transmission mechanism in transformed cells in response to the ECM-F-actin pathway in vitro. Shear stress was observed to promote the expression of complement regulatory protein CD59, but not CD46 or CD55, on EPCs. In addition, the shear stress-induced CD59 expression was confirmed to be associated with the ECM components and was alleviated in EPCs pretreated with GRGDSP, which inhibits ECM components-integrin interaction. Furthermore, shear stress also promotes the rearrangement and polymerization of F-actin. However, shear stress-induced CD59 expression was reduced when the F-actin stress fiber formation process was delayed by Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP) or destroyed by cytochalasin D (Cyto D), while Jasplakinolide (JAS) reversed the expression of CD59 through promotion of F-actin polymerization and its stabilizing capacities. Our results indicates that shear stress is an important mediator in EPC expression of CD59 regulated by the ECM-F-actin pathway, which is a key factor in preventing membrane attack complex (MAC) -mediated cell autolysis.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Actinas/genética , Antígenos CD59/genética , Células Progenitoras Endoteliales/metabolismo , Integrina alfaVbeta3/genética , Mecanotransducción Celular , Citoesqueleto de Actina/efectos de los fármacos , Citoesqueleto de Actina/ultraestructura , Actinas/metabolismo , Antígenos CD55/genética , Antígenos CD55/metabolismo , Antígenos CD59/metabolismo , Complejo de Ataque a Membrana del Sistema Complemento/efectos de los fármacos , Citocalasina D/farmacología , Depsipéptidos/farmacología , Células Progenitoras Endoteliales/citología , Células Progenitoras Endoteliales/efectos de los fármacos , Matriz Extracelular/química , Matriz Extracelular/efectos de los fármacos , Matriz Extracelular/metabolismo , Sangre Fetal/citología , Sangre Fetal/efectos de los fármacos , Sangre Fetal/metabolismo , Regulación de la Expresión Génica , Humanos , Integrina alfaVbeta3/metabolismo , Proteína Cofactora de Membrana/genética , Proteína Cofactora de Membrana/metabolismo , Oligopéptidos/farmacología , Cultivo Primario de Células , Estrés MecánicoRESUMEN
BACKGROUND: Intravenous (IV) iron preparations are widely used in the treatment of anemia in patients undergoing hemodialysis (HD). All IV iron preparations carry a risk of causing hypersensitivity reactions. However, the pathophysiological mechanism is poorly understood. We hypothesize that a relevant number of these reactions are mediated by complement activation, resulting in a pseudo-anaphylactic clinical picture known as complement activation-related pseudo allergy (CARPA). METHODS: First, the in-vitro complement-activating capacity was determined for 5 commonly used IV iron preparations using functional complement assays for the 3 pathways. Additionally, the preparations were tested in an ex-vivo model using the whole blood of healthy volunteers and HD patients. Lastly, in-vivo complement activation was tested for one preparation in HD patients. RESULTS: In the in-vitro assays, iron dextran, and ferric carboxymaltose caused complement activation, which was only possible under alternative pathway conditions. Iron sucrose may interact with complement proteins, but did not activate complement in-vitro. In the ex-vivo assay, iron dextran significantly induced complement activation in the blood of healthy volunteers and HD patients. Furthermore, in the ex-vivo assay, ferric carboxymaltose and iron sucrose only caused significant complement activation in the blood of HD patients. No in-vitro or ex-vivo complement activation was found for ferumoxytol and iron isomaltoside. IV iron therapy with ferric carboxymaltose in HD patients did not lead to significant in-vivo complement activation. CONCLUSION: This study provides evidence that iron dextran and ferric carboxymaltose have complement-activating capacities in-vitro, and hypersensitivity reactions to these drugs could be CARPA-mediated.
Asunto(s)
Anemia Ferropénica/tratamiento farmacológico , Activación de Complemento/efectos de los fármacos , Hematínicos/farmacología , Compuestos de Hierro/farmacología , Fallo Renal Crónico/terapia , Administración Intravenosa , Anemia Ferropénica/complicaciones , Complemento C1q/efectos de los fármacos , Complemento C1q/metabolismo , Complemento C3d/efectos de los fármacos , Complemento C3d/metabolismo , Complejo de Ataque a Membrana del Sistema Complemento/efectos de los fármacos , Complejo de Ataque a Membrana del Sistema Complemento/metabolismo , Disacáridos/farmacología , Disacáridos/uso terapéutico , Compuestos Férricos/farmacología , Compuestos Férricos/uso terapéutico , Sacarato de Óxido Férrico , Óxido Ferrosoférrico/farmacología , Óxido Ferrosoférrico/uso terapéutico , Ácido Glucárico/farmacología , Ácido Glucárico/uso terapéutico , Hematínicos/uso terapéutico , Humanos , Técnicas In Vitro , Compuestos de Hierro/uso terapéutico , Complejo Hierro-Dextran/farmacología , Complejo Hierro-Dextran/uso terapéutico , Fallo Renal Crónico/complicaciones , Maltosa/análogos & derivados , Maltosa/farmacología , Maltosa/uso terapéutico , Lectina de Unión a Manosa/efectos de los fármacos , Lectina de Unión a Manosa/metabolismo , Properdina/efectos de los fármacos , Properdina/metabolismo , Diálisis RenalRESUMEN
Thrombus formation leading to vaso-occlusive events is a major cause of death, and involves complex interactions between coagulation, fibrinolytic and innate immune systems. Leukocyte recruitment is a key step, mediated partly by chemotactic complement activation factors C3a and C5a. However, mechanisms mediating C3a/C5a generation during thrombosis have not been studied. In a murine venous thrombosis model, levels of thrombin-antithrombin complexes poorly correlated with C3a and C5a, excluding a central role for thrombin in C3a/C5a production. However, clot weight strongly correlated with C5a, suggesting processes triggered during thrombosis promote C5a generation. Since thrombosis elicits fibrinolysis, we hypothesized that plasmin activates C5 during thrombosis. In vitro, the catalytic efficiency of plasmin-mediated C5a generation greatly exceeded that of thrombin or factor Xa, but was similar to the recognized complement C5 convertases. Plasmin-activated C5 yielded a functional membrane attack complex (MAC). In an arterial thrombosis model, plasminogen activator administration increased C5a levels. Overall, these findings suggest plasmin bridges thrombosis and the immune response by liberating C5a and inducing MAC assembly. These new insights may lead to the development of strategies to limit thrombus formation and/or enhance resolution.
Asunto(s)
Arterias/inmunología , Complemento C5a/inmunología , Fibrinolisina/inmunología , Trombosis de la Vena/inmunología , Animales , Antitrombina III/efectos de los fármacos , Antitrombina III/inmunología , Arterias/efectos de los fármacos , Arterias/patología , Activación de Complemento/efectos de los fármacos , Activación de Complemento/inmunología , Complemento C3a/biosíntesis , Complemento C3a/inmunología , Complemento C5a/biosíntesis , Complejo de Ataque a Membrana del Sistema Complemento/efectos de los fármacos , Complejo de Ataque a Membrana del Sistema Complemento/inmunología , Factor Xa/inmunología , Factor Xa/metabolismo , Fibrinolisina/metabolismo , Humanos , Ratones , Péptido Hidrolasas/efectos de los fármacos , Péptido Hidrolasas/inmunología , Activadores Plasminogénicos/administración & dosificación , Trombina/inmunología , Trombina/metabolismo , Trombosis de la Vena/tratamiento farmacológico , Trombosis de la Vena/patologíaRESUMEN
Complement system, a major component of the natural immunity, has been recently identified as an important mediator of the dentin-pulp regeneration process through STRO-1 pulp cell recruitment by the C5a active fragment. Moreover, it has been shown recently that under stimulation with lipoteichoic acid, a complex component of the Gram-positive bacteria cell wall, human pulp fibroblasts are able to synthesize all proteins required for complement activation. However, Gram-negative bacteria, which are also involved in tooth decay, are known as powerful activators of complement system and inflammation. Here, we investigated the role of Gram-negative bacteria-induced complement activation on the pulp progenitor cell recruitment using lipopolysaccharide (LPS), a major component of all Gram-negative bacteria. Our results show that incubating pulp fibroblasts with LPS induced membrane attack complex formation and C5a release in serum-free fibroblast cultures. The produced C5a binds to the pulp progenitor cells' membrane and induces their migration toward the LPS stimulation chamber, as revealed by the dynamic transwell migration assays. The inhibition of this migration by the C5aR-specific antagonist W54011 indicates that the pulp progenitor migration is mediated by the interaction between C5a and C5aR. Our findings demonstrate, for the first time, a direct interaction between the recruitment of progenitor pulp cells and the activation of complement system generated by pulp fibroblast stimulation with LPS.
Asunto(s)
Quimiotaxis/efectos de los fármacos , Activación de Complemento/efectos de los fármacos , Complemento C5a/efectos de los fármacos , Pulpa Dental/citología , Lipopolisacáridos/farmacología , Células Madre/efectos de los fármacos , Compuestos de Anilina/farmacología , Técnicas de Cultivo de Célula , Factores Quimiotácticos/antagonistas & inhibidores , Factores Quimiotácticos/inmunología , Complemento C5a/antagonistas & inhibidores , Complemento C5a/inmunología , Complejo de Ataque a Membrana del Sistema Complemento/efectos de los fármacos , Caries Dental/microbiología , Pulpa Dental/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Fibroblastos/inmunología , Bacterias Gramnegativas/fisiología , Humanos , Factores Inmunológicos/antagonistas & inhibidores , Factores Inmunológicos/inmunología , Receptor de Anafilatoxina C5a/antagonistas & inhibidores , Tetrahidronaftalenos/farmacologíaRESUMEN
The Gram-positive bacterium Streptococcus pneumoniae is a major human pathogen that causes infections ranging from acute otitis media to life-threatening invasive disease. Pneumococci have evolved several strategies to circumvent the host immune response, in particular the complement attack. The pneumococcal glycolytic enzyme phosphoglycerate kinase (PGK) is both secreted and bound to the bacterial surface and simultaneously binds plasminogen and its tissue plasminogen activator tPA. In the present study we demonstrate that PGK has an additional role in modulating the complement attack. PGK interacted with the membrane attack complex (MAC) components C5, C7, and C9, thereby blocking the assembly and membrane insertion of MAC resulting in significant inhibition of the hemolytic activity of human serum. Recombinant PGK interacted in a dose-dependent manner with these terminal pathway proteins, and the interactions were ionic in nature. In addition, PGK inhibited C9 polymerization both in the fluid phase and on the surface of sheep erythrocytes. Interestingly, PGK bound several MAC proteins simultaneously. Although C5 and C7 had partially overlapping binding sites on PGK, C9 did not compete with either one for PGK binding. Moreover, PGK significantly inhibited MAC deposition via both the classical and alternative pathway at the pneumococcal surface. Additionally, upon activation plasmin(ogen) bound to PGK cleaved the central complement protein C3b thereby further modifying the complement attack. In conclusion, our data demonstrate for the first time to our knowledge a novel pneumococcal inhibitor of the terminal complement cascade aiding complement evasion by this important pathogen.
Asunto(s)
Proteínas Bacterianas/metabolismo , Inactivadores del Complemento/metabolismo , Complejo de Ataque a Membrana del Sistema Complemento/metabolismo , Fosfoglicerato Quinasa/metabolismo , Streptococcus pneumoniae/enzimología , Animales , Proteínas Bacterianas/genética , Activación de Complemento/efectos de los fármacos , Complemento C3b/metabolismo , Complemento C5/metabolismo , Complemento C7/metabolismo , Complemento C9/metabolismo , Inactivadores del Complemento/farmacología , Complejo de Ataque a Membrana del Sistema Complemento/efectos de los fármacos , Proteínas del Sistema Complemento/metabolismo , Relación Dosis-Respuesta a Droga , Eritrocitos/metabolismo , Citometría de Flujo , Hemólisis , Humanos , Fosfoglicerato Quinasa/genética , Plasminógeno/metabolismo , Polimerizacion/efectos de los fármacos , Unión Proteica , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , Suero/metabolismo , Ovinos , Streptococcus pneumoniae/genéticaRESUMEN
Aberrant complement activation is known to exacerbate the pathology in a spectrum of degenerative diseases of aging. We previously reported that aurin tricarboxylic acid (ATA) is an orally effective agent which prevents formation of the membrane attack complex of complement. It inhibits C9 attachment to tissue bound C5b678 and thus prevents bystander lysis of host cells. In this study, we investigated the effects of ATA on the alternative complement pathway. We found that ATA prevented cleavage of the tissue bound properdin-C3b-Factor B complex into the active C3 convertase enzyme properdin-C3b-Factor Bb. This inhibition was reversed by adding Factor D to the serum. Using enzyme-linked immunosorbent type assays, we established that ATA binds directly to Factor D and C9 but not to properdin or other complement proteins. We conclude that ATA, by inhibiting at two stages of the alternative pathway, might be a particularly effective therapeutic agent in conditions such as macular degeneration, paroxysmal nocturnal hemoglobinemia, and rheumatoid arthritis, in which activation of the alternative complement pathway initiates self damage.
Asunto(s)
Ácido Aurintricarboxílico/farmacología , Activación de Complemento/inmunología , Convertasas de Complemento C3-C5/inmunología , Complemento C9/inmunología , Complejo de Ataque a Membrana del Sistema Complemento/inmunología , Activación de Complemento/efectos de los fármacos , Complejo de Ataque a Membrana del Sistema Complemento/efectos de los fármacos , Humanos , Fármacos Neuroprotectores/farmacología , Unión ProteicaRESUMEN
Atypical hemolytic uremic syndrome (aHUS) is a rare, lifethreatening, chronic, genetic disease of uncontrolled alternative pathway complement activation. The understanding of the pathophysiology and genetics of this disease has expanded over recent decades and promising new developments in the management of aHUS have emerged. Regardless of the cause of aHUS, with or without a demonstrated mutation or autoantibody, blockade of terminal complement activation through C5 is of high interest as a mechanism to ameliorate the disease. Eculizumab, an existing monoclonal antibody directed against C5 with high affinity, prevents the perpetuation of the downstream activation of the complement cascade and the damage caused by generation of the anaphylotoxin C5a and the membrane attack complex C5b-9, by blocking C5 cleavage. We report the successful use of eculizumab in a patient after kidney transplantation and discuss the disease aHUS.
Asunto(s)
Anticuerpos Monoclonales Humanizados/uso terapéutico , Complejo de Ataque a Membrana del Sistema Complemento/inmunología , Síndrome Hemolítico-Urémico/diagnóstico , Trasplante de Riñón/efectos adversos , Síndrome Hemolítico Urémico Atípico , Complejo de Ataque a Membrana del Sistema Complemento/efectos de los fármacos , Femenino , Síndrome Hemolítico-Urémico/tratamiento farmacológico , Síndrome Hemolítico-Urémico/inmunología , Humanos , Lactante , Resultado del TratamientoRESUMEN
We have shown previously that cytokines IL-4 and IL-13 induce protection in porcine vascular endothelial cells (EC) against killing by the membrane attack complex (MAC) of human complement. This protection is intrinsic, not due to changes in complement regulatory proteins, and requires activation of Akt and sterol receptor element binding protein-1 (SREBP-1), which regulates fatty acid and phospholipid synthesis. Here we report that, compared to EC incubated in medium, IL-4-treated EC had a profound reduction in complement-mediated ATP loss and in killing assessed by vital dye uptake, but only a slight reduction in permeability disruption measured by calcein release. While controls exposed to complement lost mitochondrial membrane potential and subsequently died, protected EC maintained mitochondrial morphology and membrane potential, and remained alive. SREBP-1 and fatty acid synthase activation were required for protection and fatty acid and phospholipid synthesis, including cardiolipin, were increased after IL-4 stimulation, without increase in cholesterol content or cell proliferation. IL-4 also induced protection of EC from killing by the channel forming protein melittin, similar to protection observed for the MAC. We conclude that IL-4 induced activation of Akt/SREBP-1/lipid biosynthesis in EC, resulting in protection against MAC and melittin, in association with mitochondrial protection.
Asunto(s)
Proteínas del Sistema Complemento/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Interleucina-4/farmacología , Lípidos/biosíntesis , Meliteno/efectos adversos , Transducción de Señal/efectos de los fármacos , Animales , Western Blotting , Permeabilidad de la Membrana Celular , Separación Celular , Complejo de Ataque a Membrana del Sistema Complemento/efectos de los fármacos , Células Endoteliales/metabolismo , Células Endoteliales/ultraestructura , Citometría de Flujo , Interleucina-4/metabolismo , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Microscopía Electrónica de Transmisión , Mitocondrias/patología , Proteínas Proto-Oncogénicas c-akt/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/efectos de los fármacos , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/metabolismo , PorcinosRESUMEN
Interactions of gene therapy vectors with human blood components upon intravenous administration have a significant effect on vector efficacy and patient safety. Here we describe methods to evaluate these interactions and their effects in whole human blood, using baculovirus vectors as a model. Opsonisation of baculovirus particles by binding of IgM and C3b was demonstrated, which is likely to be the cause of the significant blood cell-associated virus that was detected. Preventing formation of the complement C5b-9 (membrane attack) complex maintained infectivity of baculovirus particles as shown by studying the effects of two specific complement inhibitors, Compstatin and a C5a receptor antagonist. Formation of macroscopic blood clots after 4h was prevented by both complement inhibitors. Pro- and anti-inflammatory cytokines Il-1beta, IL-6, IL-8 and TNF-alpha were produced at variable levels between volunteers and complement inhibitors showed patient-specific effects on cytokine levels. Whilst both complement inhibitors could play a role in protecting patients from aggressive inflammatory reactions, only Compstatin maintained virus infectivity. We conclude that this ex vivo model, used here for the first time with infectious agents, is a valuable tool in evaluating human innate immune responses to gene therapy vectors or to predict the response of individual patients as part of a clinical trial or treatment. The use of complement inhibitors for therapeutic viruses should be considered on a patient-specific basis.
Asunto(s)
Baculoviridae/inmunología , Complejo de Ataque a Membrana del Sistema Complemento/inmunología , Terapia Genética , Vectores Genéticos/sangre , Vectores Genéticos/inmunología , Inmunidad Innata , Baculoviridae/genética , Complemento C3b/inmunología , Complemento C5a/farmacología , Complejo de Ataque a Membrana del Sistema Complemento/efectos de los fármacos , Citocinas/biosíntesis , Citocinas/inmunología , Vectores Genéticos/genética , Humanos , Inmunidad Innata/efectos de los fármacos , Inmunoensayo , Inmunoglobulina M/sangre , Péptidos Cíclicos/farmacologíaRESUMEN
Guillain-Barré syndrome and its clinical variants, including the anti-GQ1b ganglioside-mediated Miller Fisher syndrome (MFS), comprise the world's leading cause of acute neuromuscular paralysis. Presently, no specific drug therapies exist. The complement cascade, which is activated in these patients, forms an attractive drug target. In this study, we tested whether the complement C5-inhibiting recombinant protein, rEV576, was able to prevent neural injury in a previously developed in vitro mouse model for MFS. Mouse hemidiaphragm preparations were treated with anti-GQ1b antibody and normal human serum as a source of complement with added rEV576 or control protein. Immunohistology in control tissue showed deposition of C3c and membrane attack complex at neuromuscular junctions (NMJs), along with terminal motor axonal neurofilament degradation as well as ethidium homodimer-2 staining showing perisynaptic Schwann cell (pSC) injury. Electrophysiological and functional analyses showed block of synaptic transmission at the NMJ after an initial period of a dramatically high level of asynchronous acetylcholine release. In tissue treated with rEV576, all these indicators of motor neuronal damage were absent, except for the presence of C3c, indicating effective inhibition of C5. These results demonstrate that rEV576 effectively prevents development of neuronal and pSC damage in experimental murine neuropathy.
Asunto(s)
Complemento C5/antagonistas & inhibidores , Proteínas Inactivadoras de Complemento/farmacología , Complejo de Ataque a Membrana del Sistema Complemento/efectos de los fármacos , Síndrome de Miller Fisher/tratamiento farmacológico , Unión Neuromuscular/efectos de los fármacos , Animales , Diafragma/efectos de los fármacos , Modelos Animales de Enfermedad , Potenciales Evocados/efectos de los fármacos , Técnica del Anticuerpo Fluorescente , Procesamiento de Imagen Asistido por Computador , Inmunohistoquímica , Proteínas de Insectos/farmacología , Masculino , Ratones , Ratones Endogámicos BALB C , Microscopía Confocal , Técnicas de Cultivo de Órganos , Proteínas Recombinantes/farmacologíaAsunto(s)
Anticuerpos Monoclonales/farmacología , Inflamación/terapia , Proteínas de la Membrana/inmunología , Receptores de Complemento/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales Humanizados , Convertasas de Complemento C3-C5/antagonistas & inhibidores , Convertasas de Complemento C3-C5/metabolismo , Complejo de Ataque a Membrana del Sistema Complemento/efectos de los fármacos , Complejo de Ataque a Membrana del Sistema Complemento/metabolismo , Proteínas del Sistema Complemento/inmunología , Terapias Complementarias , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Receptor de Anafilatoxina C5a , Receptores de Complemento/genética , Receptores de Complemento/metabolismoRESUMEN
The protein zero (P0) glycoprotein is an important component of compact peripheral nerve myelin produced by the glial cells of the mammalian peripheral nervous system. P0 mRNA expression is reduced following exposure of Schwann cells to sublytic C5b-9, the terminal activation complex of the complement cascade. Sublytic complement treatment decreased P0 mRNA by 81% within 6 h and required C5b-9 assembly. C5b-9 induced a threefold increase in both JNK1 activity and c-jun mRNA within 20 and 30 min, respectively, compared with cells treated with either human serum depleted of complement component C7 (C7dHS) or medium alone. Sublytic C5b-9 stimulation, in the presence of the transcription inhibitor Actinomycin D, decreased P0 mRNA expression by 52%, indicating that mRNA was selectively destabilized. This effect was prevented by pretreatment with L-JNK inhibitor 1 (L-JNKI1). To study a potential inhibition of P0 gene transcription, we transfected Schwann cells with a P0 promoter-firefly luciferase construct. Sublytic C5b-9 stimulation of the transfected cells decreased luciferase activity by 82% at 6 h, and this effect was prevented by pretreatment with L-JNKI1 inhibitor. Our results indicate that the ability of C5b-9 in vitro to affect P0 gene expression is mediated via JNK1 activation that leads to enhanced mRNA decay and transcriptional repression of P0.
Asunto(s)
Complejo de Ataque a Membrana del Sistema Complemento/metabolismo , Activación Enzimática/fisiología , Proteína Quinasa 8 Activada por Mitógenos/metabolismo , Proteína P0 de la Mielina/metabolismo , Células de Schwann/metabolismo , Animales , Northern Blotting , Complejo de Ataque a Membrana del Sistema Complemento/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Expresión Génica/fisiología , Proteína Quinasa 8 Activada por Mitógenos/efectos de los fármacos , Proteína P0 de la Mielina/efectos de los fármacos , Proteína P0 de la Mielina/genética , Estabilidad del ARN/fisiología , ARN Mensajero , Ratas , Ratas Sprague-Dawley , Células de Schwann/efectos de los fármacos , Transcripción GenéticaRESUMEN
Carotenoids are a naturally occurring group of compounds that possess antioxidant properties. Most natural carotenoids display poor aqueous solubility and tend to form aggregates in solution. Disodium disuccinate astaxanthin (DDA; Cardax) is a water-dispersible synthetic carotenoid that rapidly and preferentially associates with serum albumin, thereby preventing the formation of supramolecular complexes and facilitating its efficacy after parenteral administration. This study investigated the ability of DDA to reduce inflammation and myocardial injury in a rabbit model of ischemia/reperfusion. DDA (50 mg/kg/day) or saline was administered i.v. for 4 consecutive days before the initiation of the protocol for induction of myocardial ischemia/reperfusion. On the 5th day, rabbits underwent 30 min of coronary artery occlusion, followed by a 3-h reperfusion period. Myocardial infarct size, as a percentage of the area at risk, was calculated for both groups. Infarct size was 52.5 +/- 7.5% in the vehicle-treated (n = 9) and 25.8 +/- 4.7% in the DDA-treated (n = 9) animals (p < 0.01 versus vehicle; mean myocardial salvage = 51%). To evaluate the anti-inflammatory effects of DDA, complement activity was assessed at the end of reperfusion using a red blood cell lysis assay. DDA administration significantly reduced (p < 0.01) the activation of the complement system in the serum. The current results, coupled with the well established antioxidant ability of carotenoids, suggest that the mechanism(s) of action by which DDA reduces the tissue damage associated with reperfusion injury may include both antioxidant and anticomplement components.
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Adyuvantes Inmunológicos/farmacología , Activación de Complemento/efectos de los fármacos , Proteínas Inactivadoras de Complemento , Daño por Reperfusión Miocárdica/fisiopatología , beta Caroteno/análogos & derivados , Adyuvantes Inmunológicos/farmacocinética , Animales , Proteína C-Reactiva/metabolismo , Complejo de Ataque a Membrana del Sistema Complemento/efectos de los fármacos , Eritrocitos/efectos de los fármacos , Técnica del Anticuerpo Fluorescente , Hemodinámica/efectos de los fármacos , Hemólisis/efectos de los fármacos , Masculino , Infarto del Miocardio/patología , Daño por Reperfusión Miocárdica/patología , Miocardio/patología , Conejos , Sales de Tetrazolio , Tiazoles , Distribución Tisular , Troponina I/sangre , Troponina I/metabolismo , Xantófilas , beta Caroteno/farmacocinética , beta Caroteno/farmacologíaRESUMEN
Insidious attack of cortical neurons by complement has been implicated in Alzheimer's and other neurodegenerative diseases. Excitotoxicity, triggered by excessive activation of glutamate receptors, has been implicated in neuronal death following diverse insults, including ischemia and seizures. Clinical studies suggested that a minimal excitotoxic insult might sensitize neurons to complement attack. We found that fleeting activation of ionotropic glutamate receptors sensitizes neurons but not astrocytes to complement attack. The complement molecule effecting cytotoxicity was the membrane attack complex. The site within the complement cascade at which sensitization was effected was the membrane attack pathway. Sensitization mediated by glutamate receptor activation required Ca(2+)(o) and generation of reactive oxygen species. These in vitro findings predict that a fleeting excitotoxic insult could act synergistically with complement to destroy cortical neurons and accelerate neurological deterioration.
Asunto(s)
Enfermedad de Alzheimer/inmunología , Membrana Celular/inmunología , Corteza Cerebral/inmunología , Complejo de Ataque a Membrana del Sistema Complemento/inmunología , Neuronas/inmunología , Receptores de Glutamato/inmunología , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/fisiopatología , Animales , Astrocitos/efectos de los fármacos , Astrocitos/inmunología , Astrocitos/metabolismo , Señalización del Calcio/efectos de los fármacos , Señalización del Calcio/inmunología , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Células Cultivadas , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/metabolismo , Técnicas de Cocultivo , Complejo de Ataque a Membrana del Sistema Complemento/efectos de los fármacos , Complejo de Ataque a Membrana del Sistema Complemento/toxicidad , Relación Dosis-Respuesta a Droga , Antagonistas de Aminoácidos Excitadores/farmacología , Feto , Ácido Glutámico/toxicidad , L-Lactato Deshidrogenasa/efectos de los fármacos , L-Lactato Deshidrogenasa/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neurotoxinas/toxicidad , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/inmunología , Especies Reactivas de Oxígeno/metabolismo , Receptores de Glutamato/efectos de los fármacos , Receptores de Glutamato/metabolismoRESUMEN
OBJECTIVE: Reports evaluating the efficacy of heparin-bonded circuits to blunt inflammation, platelet dysfunction, and thrombin generation in response to cardiopulmonary bypass have varied. We hypothesized that this variability may in part be related to the use of cardiotomy suction, which has been demonstrated to reintroduce procoagulant and proinflammatory factors into the systemic circulation during cardiopulmonary bypass. A prospective, randomized study was undertaken to evaluate the specific effects of cardiotomy suction. METHODS: Thirty-six patients undergoing first-time, nonemergency coronary artery bypass grafting with cardiopulmonary bypass were randomly assigned to one of three treatment groups: group I, non-heparin-bonded circuits with the use of cardiotomy suction (n = 12); group II, Duraflo II (BCR-3500; Jostra Bentley Corp, Irvine, Calif) heparin-bonded circuits with cardiotomy suction (n = 12); and group III, Duraflo II heparin-bonded circuits without cardiotomy suction (n = 12). Thrombin generation, neutrophil activation (polymorphonuclear elastase), platelet activation (beta-thromboglobulin), and neuronal injury (neuron-specific enolase) were analyzed by enzyme-linked immunosorbent assays after cardiopulmonary bypass and compared with prebypass levels. Results are presented as mean +/- SEM. RESULTS: Prebypass levels of all markers were similar among treatment groups. However, postbypass levels were significantly and consistently highest in group I relative to groups II and III. Thrombin generation levels were 5.0 +/- 0.9 nmol/L in group I, 3.0 +/- 0.6 nmol/L in group II, and 1.5 +/- 0.1 nmol/L in group III (P <.05 vs group II and P <.001 vs group I). Polymorphonuclear elastase levels were 307 +/- 64 microg/L in group I, 128 +/- 24 microg/L in group II (P <.05 vs group I), and 75 +/- 14 microg/L in group III (P <.001 vs group I). beta-Thromboglobulin levels were 2692 +/- 401 IU/mL in group I, 912 +/- 99 IU/mL in group II (P =.001 vs group I), and 646 +/- 133 IU/mL in group III (P =.001 vs group I). Neuron-specific enolase levels were 9.8 +/- 0.9 ng/mL in group I, 10.5 +/- 1.6 ng/mL in group II, and 4.2 +/- 0.5 ng/mL in group III (P =.001 vs groups I and II). CONCLUSIONS: Use of cardiotomy suction resulted in significant increases in thrombin, neutrophil, and platelet activation, as well as the release of neuron-specific enolase, after cardiopulmonary bypass. Limiting increases in these markers would be best accomplished by eliminating cardiotomy suction and routinely using heparin-bonded circuits whenever possible.