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1.
Commun Biol ; 7(1): 649, 2024 May 27.
Artículo en Inglés | MEDLINE | ID: mdl-38802531

RESUMEN

Salivary complement inhibitors occur in many of the blood feeding arthropod species responsible for transmission of pathogens. During feeding, these inhibitors prevent the production of proinflammatory anaphylatoxins, which may interfere with feeding, and limit formation of the membrane attack complex which could damage arthropod gut tissues. Salivary inhibitors are, in many cases, novel proteins which may be pharmaceutically useful or display unusual mechanisms that could be exploited pharmaceutically. Albicin is a potent inhibitor of the alternative pathway of complement from the saliva of the malaria transmitting mosquito, Anopheles albimanus. Here we describe the cryo-EM structure of albicin bound to C3bBb, the alternative C3 convertase, a proteolytic complex that is responsible for cleavage of C3 and amplification of the complement response. Albicin is shown to induce dimerization of C3bBb, in a manner similar to the bacterial inhibitor SCIN, to form an inactive complex unable to bind the substrate C3. Size exclusion chromatography and structures determined after 30 minutes of incubation of C3b, factor B (FB), factor D (FD) and albicin indicate that FBb dissociates from the inhibited dimeric complex leaving a C3b-albicin dimeric complex which apparently decays more slowly.


Asunto(s)
Anopheles , Complemento C3b , Proteínas de Insectos , Proteínas y Péptidos Salivales , Microscopía por Crioelectrón , Proteínas y Péptidos Salivales/química , Proteínas y Péptidos Salivales/metabolismo , Complemento C3b/química , Complemento C3b/metabolismo , Modelos Químicos , Animales , Proteínas de Insectos/química , Proteínas de Insectos/metabolismo , Anopheles/química , Anopheles/clasificación , Secuencia de Aminoácidos , Humanos , Inactivadores del Complemento/química , Inactivadores del Complemento/metabolismo
2.
J Transl Med ; 22(1): 164, 2024 02 16.
Artículo en Inglés | MEDLINE | ID: mdl-38365806

RESUMEN

BACKGROUND: Pulmonary arterial hypertension (PAH) is a severe cardiopulmonary disease characterized by complement dependent and proinflammatory activation of macrophages. However, effective treatment for complement activation in PAH is lacking. We aimed to explore the effect and mechanism of CP40-KK (a newly identified analog of selective complement C3 inhibitor CP40) in the PAH model. METHODS: We used western blotting, immunohistochemistry, and immunofluorescence staining of lung tissues from the monocrotaline (MCT)-induced rat PAH model to study macrophage infiltration, NLPR3 inflammasome activation, and proinflammatory cytokines (IL-1ß and IL-18) release. Surface plasmon resonance (SPR), ELISA, and CH50 assays were used to test the affinity between CP40-KK and rat/human complement C3. CP40-KK group rats only received CP40-KK (2 mg/kg) by subcutaneous injection at day 15 to day 28 continuously. RESULTS: C3a was significantly upregulated in the plasma of MCT-treated rats. SPR, ELISA, and CH50 assays revealed that CP40-KK displayed similar affinity binding to human and rat complement C3. Pharmacological inhibition of complement C3 cleavage (CP40-KK) could ameliorate MCT-induced NLRP3 inflammasome activity, pulmonary vascular remodeling, and right ventricular hypertrophy. Mechanistically, increased proliferation of pulmonary arterial smooth muscle cells is closely associated with macrophage infiltration, NLPR3 inflammasome activation, and proinflammatory cytokines (IL-1ß and IL-18) release. Besides, C3a enhanced IL-1ß activity in macrophages and promoted pulmonary arterial smooth muscle cell proliferation in vitro. CONCLUSION: Our findings suggest that CP40-KK treatment was protective in the MCT-induced rat PAH model, which might serve as a therapeutic option for PAH.


Asunto(s)
Hipertensión Pulmonar , Hipertensión Arterial Pulmonar , Ratas , Humanos , Animales , Hipertensión Arterial Pulmonar/tratamiento farmacológico , Inflamasomas/metabolismo , Interleucina-18/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Complemento C3/metabolismo , Inactivadores del Complemento/efectos adversos , Inactivadores del Complemento/metabolismo , Arteria Pulmonar/metabolismo , Citocinas/metabolismo , Modelos Animales de Enfermedad
3.
Adv Ther ; 40(6): 2752-2772, 2023 06.
Artículo en Inglés | MEDLINE | ID: mdl-37072660

RESUMEN

INTRODUCTION: Hemolysis in paroxysmal nocturnal hemoglobinuria (PNH) is complement-mediated due to the lack of complement inhibitors in the hemopoietic cell membranes, making complement inhibition the best approach to manage PNH. Three complement inhibitors are approved by the European Medicines Agency as targeted therapy for PNH: eculizumab and ravulizumab, two humanized monoclonal antibodies targeting the same complement 5 (C5) epitope, approved in 2007 and 2019, respectively, and the more recently approved cyclic peptide, the complement 3 (C3) inhibitor pegcetacoplan. Although national and international PNH treatment guidelines exist, they do not take into consideration the latest clinical trial evidence. Given the lack of evidence-based data for some clinical situations encountered in real life, we identified specific populations of patients who may benefit from switching to proximal C3 from terminal C5 inhibition. METHODS: The expert recommendations presented here were created using a Delphi-like process by a group of expert PNH specialists across Central Europe. Based on an initial advisory board meeting discussion, recommendations were prepared and reviewed as part of a Delphi survey to test agreement. RESULTS: Using a systematic approach, literature databases were searched for relevant studies, and 50 articles were reviewed by the experts and included as supporting evidence. CONCLUSION: Implementation of these recommendations uniformly across healthcare institutions will promote the best use of complement inhibition in managing PNH, and has the potential to positively impact patient outcomes in Central Europe and worldwide.


Asunto(s)
Hemoglobinuria Paroxística , Humanos , Hemoglobinuria Paroxística/tratamiento farmacológico , Testimonio de Experto , Inactivadores del Complemento/uso terapéutico , Inactivadores del Complemento/metabolismo , Complemento C3/metabolismo , Complemento C3/uso terapéutico , Complemento C5/uso terapéutico , Europa (Continente)
4.
Antioxid Redox Signal ; 37(10-12): 781-801, 2022 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-34806406

RESUMEN

Significance: The metabolic disorder, diabetes mellitus, results in microvascular complications, including diabetic kidney disease (DKD), which is partly believe to involve disrupted energy generation in the kidney, leading to injury that is characterized by inflammation and fibrosis. An increasing body of evidence indicates that the innate immune complement system is involved in the pathogenesis of DKD; however, the precise mechanisms remain unclear. Recent Advances: Complement, traditionally thought of as the prime line of defense against microbial intrusion, has recently been recognized to regulate immunometabolism. Studies have shown that the complement activation products, Complement C5a and C3a, which are potent pro-inflammatory mediators, can mediate an array of metabolic responses in the kidney in the diabetic setting, including altered fuel utilization, disrupted mitochondrial respiratory function, and reactive oxygen species generation. In diabetes, the lectin pathway is activated via autoreactivity toward altered self-surfaces known as danger-associated molecular patterns, or via sensing altered carbohydrate and acetylation signatures. In addition, endogenous complement inhibitors can be glycated, whereas diet-derived glycated proteins can themselves promote complement activation, worsening DKD, and lending support for environmental influences as an additional avenue for propagating complement-induced inflammation and kidney injury. Critical Issues: Recent evidence indicates that conventional renoprotective agents used in DKD do not target the complement, leaving this web of inflammatory stimuli intact. Future Directions: Future studies should focus on the development of novel pharmacological agents that target the complement pathway to alleviate inflammation, oxidative stress, and kidney fibrosis, thereby reducing the burden of microvascular diseases in diabetes. Antioxid. Redox Signal. 37, 781-801.


Asunto(s)
Diabetes Mellitus , Nefropatías Diabéticas , Carbohidratos , Complemento C5a/metabolismo , Inactivadores del Complemento/metabolismo , Diabetes Mellitus/metabolismo , Nefropatías Diabéticas/etiología , Nefropatías Diabéticas/metabolismo , Fibrosis , Humanos , Inflamación/metabolismo , Mediadores de Inflamación/metabolismo , Riñón/metabolismo , Lectinas/metabolismo , Especies Reactivas de Oxígeno/metabolismo
5.
Semin Immunopathol ; 43(6): 799-816, 2021 12.
Artículo en Inglés | MEDLINE | ID: mdl-34191092

RESUMEN

The complement system is an important defense mechanism against pathogens; however, in certain pathologies, the system also attacks human cells, such as red blood cells (RBCs). In paroxysmal nocturnal hemoglobinuria (PNH), RBCs lack certain complement regulators which sensitize them to complement-mediated lysis, while in autoimmune hemolytic anemia (AIHA), antibodies against RBCs may initiate complement-mediated hemolysis. In recent years, complement inhibition has improved treatment prospects for these patients, with eculizumab now the standard of care for PNH patients. Current complement inhibitors are however not sufficient for all patients, and they come with high costs, patient burden, and increased infection risk. This review gives an overview of the underlying pathophysiology of complement-mediated hemolysis in PNH and AIHA, the role of therapeutic complement inhibition nowadays, and the high number of complement inhibitors currently under investigation, as for almost every complement protein, an inhibitor is being developed. The focus lies with novel therapeutics that inhibit complement activity specifically in the pathway that causes pathology or those that reduce costs or patient burden through novel administration routes.


Asunto(s)
Hemoglobinuria Paroxística , Inactivadores del Complemento/metabolismo , Inactivadores del Complemento/farmacología , Inactivadores del Complemento/uso terapéutico , Proteínas del Sistema Complemento/metabolismo , Eritrocitos/metabolismo , Eritrocitos/patología , Hemoglobinuria Paroxística/tratamiento farmacológico , Hemoglobinuria Paroxística/etiología , Hemólisis , Humanos
6.
J Biol Chem ; 296: 100083, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33199367

RESUMEN

Inhibition of the alternative pathway (AP) of complement by saliva from Anopheles mosquitoes facilitates feeding by blocking production of the anaphylatoxins C3a and C5a, which activate mast cells leading to plasma extravasation, pain, and itching. We have previously shown that albicin, a member of the SG7 protein family from An. Albimanus, blocks the AP by binding to and inhibiting the function of the C3 convertase, C3bBb. Here we show that SG7.AF, the albicin homolog from An. freeborni, has a similar potency to albicin but is more active in the presence of properdin, a plasma protein that acts to stabilize C3bBb. Conversely, albicin is highly active in the absence or presence of properdin. Albicin and SG7.AF stabilize the C3bBb complex in a form that accumulates on surface plasmon resonance (SPR) surfaces coated with properdin, but SG7.AF binds with lower affinity than albicin. Albicin induces oligomerization of the complex in solution, suggesting that it is oligomerization that leads to stabilization on SPR surfaces. Anophensin, the albicin ortholog from An. stephensi, is only weakly active as an inhibitor of the AP, suggesting that the SG7 family may play a different functional role in this species and other species of the subgenus Cellia, containing the major malaria vectors in Africa and Asia. Crystal structures of albicin and SG7.AF reveal a novel four-helix bundle arrangement that is stabilized by an N-terminal hydrogen bonding network. These structures provide insight into the SG7 family and related mosquito salivary proteins including the platelet-inhibitory 30 kDa family.


Asunto(s)
Inactivadores del Complemento/química , Inactivadores del Complemento/metabolismo , Properdina/metabolismo , Saliva/química , Animales , Anopheles , Convertasas de Complemento C3-C5/genética , Convertasas de Complemento C3-C5/metabolismo , Vía Alternativa del Complemento/genética , Vía Alternativa del Complemento/fisiología , Cristalografía por Rayos X , Culicidae , Proteínas de Insectos/genética , Proteínas de Insectos/metabolismo , Properdina/genética , Resonancia por Plasmón de Superficie
7.
JCI Insight ; 5(13)2020 07 09.
Artículo en Inglés | MEDLINE | ID: mdl-32641582

RESUMEN

Multiple organ failure in sepsis is a progressive failure of several interdependent organ systems. Liver dysfunction occurs early during sepsis and is directly associated with patient death; however, the underlying mechanism of liver dysfunction is unclear. Platelet transfusion benefits patients with sepsis, and inhibition of complement activation protects liver function in septic animals. Herein, we explored the potential link between platelets, complement activation, and liver dysfunction in sepsis. We found that deletion of platelet C-type lectin-like receptor 2 (CLEC-2) exacerbated liver dysfunction in early sepsis. Platelet CLEC-2-deficient mice exhibited higher complement activation, more severe complement attack in the liver, and lower plasma levels of complement inhibitors at early time points after E. coli infection. Circulating monocytes expressed the CLEC-2 ligand podoplanin in early sepsis, and podoplanin binding induced release of complement inhibitors from platelets. Injection of complement inhibitors released from platelets reduced complement attack and attenuated liver dysfunction in septic mice. These findings indicate a new function of platelets in the regulation of complement activation during sepsis.


Asunto(s)
Inactivadores del Complemento/farmacología , Hígado/efectos de los fármacos , Glicoproteínas de Membrana/metabolismo , Monocitos/metabolismo , Sepsis/complicaciones , Animales , Plaquetas/metabolismo , Inactivadores del Complemento/metabolismo , Hígado/metabolismo , Glicoproteínas de Membrana/efectos de los fármacos , Glicoproteínas de Membrana/genética , Ratones , Monocitos/efectos de los fármacos , Activación Plaquetaria/efectos de los fármacos , Activación Plaquetaria/fisiología , Sepsis/inducido químicamente , Regulación hacia Arriba
8.
Expert Opin Biol Ther ; 20(9): 991-998, 2020 09.
Artículo en Inglés | MEDLINE | ID: mdl-32602752

RESUMEN

INTRODUCTION: Acetylcholine receptor antibody-positive generalized myasthenia gravis (gMG) is effectively treated with symptomatic and immunosuppressive drugs but a proportion of patients has a persistent disease and severe adverse events (AEs). The unmet medical needs are specific immunosuppression and AE lowering. Eculizumab blocks C5 protecting neuromuscular junction from the destructive autoantibody effects. Phase II (Study C08-001) and III (ECU-MG-301) studies, with the open-label extension (ECU-MG-302), demonstrated eculizumab efficacy and safety in refractory gMG patients. AREAS COVERED: We provide an overview of eculizumab biological features, clinical efficacy, and safety in gMG patients, highlighting our perspective on the drug positioning in the MG treatment algorithm. EXPERT OPINION: Eculizumab has the potential to significantly change the immunosuppressive approach in gMG offering the opportunity to avoid or delay corticosteroids' use due to its speed and selective mechanism of action. Eculizumab prescription will depend on: 1. ability to modify the natural disease course; 2. sustainability in the clinical practice (cost/effectiveness ratio); 3. drug-induced AE reduction. At present we are missing a controlled study on its use as a first-line treatment. We think that immunosuppression in MG will change significantly in the next years by adopting more focused 'Precision Medicine' approaches, and Eculizumab seems to satisfy such a promise.


Asunto(s)
Anticuerpos Monoclonales Humanizados/uso terapéutico , Inactivadores del Complemento/uso terapéutico , Miastenia Gravis/tratamiento farmacológico , Anticuerpos Monoclonales Humanizados/inmunología , Anticuerpos Monoclonales Humanizados/metabolismo , Ensayos Clínicos como Asunto , Complemento C5/inmunología , Inactivadores del Complemento/inmunología , Inactivadores del Complemento/metabolismo , Semivida , Humanos , Miastenia Gravis/patología , Resultado del Tratamiento
9.
Mol Vis ; 26: 370-377, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32476817

RESUMEN

Purpose: Risk for age-related macular degeneration (AMD), a slowly progressing, complex disease, is tied to an overactive complement system. Efforts are under way to develop an anticomplement-based treatment to be delivered locally or systemically. We developed an alternative pathway (AP) inhibitor fusion protein consisting of a complement receptor-2 fragment linked to the inhibitory domain of factor H (CR2-fH), which reduces the size of mouse choroidal neovascularization (CNV) when delivered locally or systemically. Specifically, we confirmed that ARPE-19 cells genetically engineered to produce CR2-fH reduce CNV lesion size when encapsulated and placed intravitreally. We extend this observation by delivering the encapsulated cells systemically in Matrigel. Methods: ARPE-19 cells were generated to stably express CR2 or CR2-fH, microencapsulated using sodium alginate, and injected subcutaneously in Matrigel into 2-month-old C57BL/6J mice. Four weeks after implantation, CNV was induced using argon laser photocoagulation. Progression of CNV was analyzed using optical coherence tomography. Bioavailability of CR2-fH was evaluated in Matrigel plugs with immunohistochemistry, as well as in ocular tissue with dot blots. Efficacy as an AP inhibitor was confirmed with protein chemistry. Results: An efficacious number of implanted capsules to reduce CNV was identified. Expression of the fusion protein systemically did not elicit an immune response. Bioavailability studies showed that CR2-fH was present in the RPE/choroid fractions of the treated mice, and reduced CNV-associated ocular complement activation. Conclusions: These findings indicate that systemic production of the AP inhibitor CR2-fH can reduce CNV in the mouse model.


Asunto(s)
Cápsulas/química , Encapsulación Celular/métodos , Neovascularización Coroidal/metabolismo , Neovascularización Coroidal/terapia , Colágeno/química , Factor H de Complemento/antagonistas & inhibidores , Inactivadores del Complemento/farmacología , Laminina/química , Proteoglicanos/química , Animales , Disponibilidad Biológica , Línea Celular , Factor H de Complemento/metabolismo , Inactivadores del Complemento/metabolismo , Combinación de Medicamentos , Expresión Génica , Humanos , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Microscopía Fluorescente , Dominios Proteicos , Receptores de Complemento 3d/genética , Receptores de Complemento 3d/metabolismo , Proteínas Recombinantes , Tomografía de Coherencia Óptica
10.
FASEB J ; 34(5): 6598-6612, 2020 05.
Artículo en Inglés | MEDLINE | ID: mdl-32219899

RESUMEN

Inhibition of the complement activation has emerged as an option for treatment of a range of diseases. Activation of the lectin and alternative pathways (LP and AP, respectively) contribute to the deterioration of conditions in certain diseases such as ischemia-reperfusion injuries and age-related macular degeneration (AMD). In the current study, we generated dual complement inhibitors of the pathways MAp44-FH and sMAP-FH by fusing full-length MAp44 or small mannose-binding lectin-associated protein (sMAP), LP regulators, with the N-terminal five short consensus repeat (SCR) domains of complement factor H (SCR1/5-FH), an AP regulator. The murine forms of both fusion proteins formed a complex with endogenous mannose-binding lectin (MBL) or ficolin A in the circulation when administered in mice intraperitoneally. Multiple complement activation assays revealed that sMAP-FH had significantly higher inhibitory effects on activation of the LP and AP in vivo as well as in vitro compared to MAp44-FH. Human form of sMAP-FH also showed dual inhibitory effects on LP and AP activation in human sera. Our results indicate that the novel fusion protein sMAP-FH inhibits both the LP and AP activation in mice and in human sera, and could be an effective therapeutic agent for diseases in which both the LP and AP activation are significantly involved.


Asunto(s)
Inactivadores del Complemento/metabolismo , Vía Alternativa del Complemento/inmunología , Lectinas/inmunología , Lectina de Unión a Manosa/metabolismo , Serina Proteasas Asociadas a la Proteína de Unión a la Manosa/metabolismo , Animales , Activación de Complemento/inmunología , Factor H de Complemento/inmunología , Factor H de Complemento/metabolismo , Inactivadores del Complemento/inmunología , Femenino , Humanos , Lectinas/metabolismo , Lectina de Unión a Manosa/inmunología , Serina Proteasas Asociadas a la Proteína de Unión a la Manosa/inmunología , Ratones , Ratones Endogámicos C57BL
11.
Expert Opin Biol Ther ; 20(3): 227-237, 2020 03.
Artículo en Inglés | MEDLINE | ID: mdl-32011183

RESUMEN

Introduction: Eculizumab, which is indicated to treat patients with paroxysmal nocturnal hemoglobinuria (PNH), is a life-changing, life-saving therapy that decreases intravascular hemolysis and thrombosis and improves survival. Some eculizumab-treated patients, however, experience breakthrough hemolysis; and overall, the burden of the treatment schedule (intravenous infusions every 2 weeks) is substantial. Ravulizumab is a long-acting, second-generation complement component 5 (C5) inhibitor that is administered intravenously every 8 weeks. It is approved in the United States (December 2018), Japan (June 2019), Europe (July 2019), and Canada and Brazil (September 2019).Areas covered: This article reviews data presented in journal articles identified on Medline/PubMed, abstracts presented at hematology meetings, and information posted on ClinicalTrials.gov and Alexion.com. Emphasis is placed on the non-inferiority of ravulizumab compared to eculizumab and the advantages of the 8-week, weight-based, dosing regimen.Expert opinion: In phase 3 trials, ravulizumab has been shown to be as safe and efficacious as eculizumab, to be associated numerically with lower rates of breakthrough hemolysis (p for non-inferiority <0.0004), and to be preferred over eculizumab by most patients. Ravulizumab is likely to replace eculizumab as the first-line treatment for PNH both in patients who are naive to eculizumab treatment and in patients who are clinically stable on eculizumab.


Asunto(s)
Anticuerpos Monoclonales Humanizados/uso terapéutico , Inactivadores del Complemento/uso terapéutico , Hemoglobinuria Paroxística/tratamiento farmacológico , Anticuerpos Monoclonales Humanizados/efectos adversos , Anticuerpos Monoclonales Humanizados/química , Inactivadores del Complemento/efectos adversos , Inactivadores del Complemento/metabolismo , Esquema de Medicación , Cefalea/etiología , Hemólisis , Humanos , Inyecciones Intraventriculares , Calidad de Vida , Resultado del Tratamiento
12.
BMC Nephrol ; 20(1): 307, 2019 08 07.
Artículo en Inglés | MEDLINE | ID: mdl-31390992

RESUMEN

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 , Recurrencia
13.
AAPS J ; 20(6): 106, 2018 10 02.
Artículo en Inglés | MEDLINE | ID: mdl-30280286

RESUMEN

Complement protein C5a is recognized as an important component of the alternative complement pathway. Its role is prominent enough to garner interest not only as a biomarker, but also as a potential therapeutic target. Bioanalytical challenges have been posed in proper quantitation of free C5a due to interference from its precursor, C5. Additionally, free therapeutic target quantitation can be difficult due to effects of sample dilution and prolonged sample incubation when therapeutic is used as capture reagent. Gyrolab technology enables quantitation of free target analyte with minimal sample dilution and rapid sample incubations, thus enabling in vitro results that are more representative of in vivo pharmacodynamics. When coupled with strategic sample pretreatment, Gyrolab offers an opportunity to quantitate free C5a in human plasma with an assay that vastly diminishes C5 interference. A Gyrolab assay for the quantitation of free C5a in human plasma was developed and validated. Validation results confirmed that proper sample pretreatment and use of the Gyrolab platform yield accurate and reliable results. Due to the advantages that it provides, Gyrolab has become our default technology of choice for quantitation of free target.


Asunto(s)
Métodos Analíticos de la Preparación de la Muestra/métodos , Bioensayo/métodos , Complemento C5a/análisis , Anticuerpos Monoclonales Humanizados/inmunología , Anticuerpos Monoclonales Humanizados/metabolismo , Bioensayo/instrumentación , Complemento C5a/inmunología , Complemento C5a/metabolismo , Inactivadores del Complemento/inmunología , Inactivadores del Complemento/metabolismo , Humanos , Inmunoensayo/instrumentación , Inmunoensayo/métodos , Límite de Detección
14.
Infect Immun ; 86(8)2018 08.
Artículo en Inglés | MEDLINE | ID: mdl-29784858

RESUMEN

Immune modulators are known to be produced by matured biofilms and during different stages of planktonic growth of Staphylococcus aureus Little is known about immune modulator production during the early stages of biofilm formation, thus raising the following question: how does S. aureus protect itself from the innate immune responses at these stages? Therefore, we determined the production of the following immune modulators: chemotaxis inhibitory protein of staphylococci (CHIPS); staphylococcal complement inhibitor (SCIN); formyl peptide receptor-like 1 inhibitor; gamma-hemolysin component B; leukocidins D, E, and S; staphylococcal superantigen-like proteins 1, 3, 5, and 9; and staphylococcal enterotoxin A. Production was determined during in vitro biofilm formation in Iscove's modified Dulbecco's medium at different time points using a competitive Luminex assay and mass spectrometry. Both methods demonstrated the production of the immune modulators SCIN and CHIPS during the early stages of biofilm formation. The green fluorescence protein promoter fusion technology confirmed scn (SCIN) and, to a lesser extent, chp (CHIPS) transcription during the early stages of biofilm formation. Furthermore, we found that SCIN could inhibit human complement activation induced by early biofilms, indicating that S. aureus is able to modulate the innate immune system already during the early stages of biofilm formation in vitro These results form a stepping stone toward elucidating the role of immune modulators in the establishment of biofilms in vivo and present opportunities to develop preventive strategies.


Asunto(s)
Biopelículas/crecimiento & desarrollo , Inactivadores del Complemento/metabolismo , Factores Inmunológicos/metabolismo , Staphylococcus aureus/crecimiento & desarrollo , Activación de Complemento , Medios de Cultivo , Perfilación de la Expresión Génica , Humanos , Inmunoensayo , Mediciones Luminiscentes , Espectrometría de Masas , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/aislamiento & purificación , Staphylococcus aureus/metabolismo
15.
Viruses ; 10(5)2018 04 25.
Artículo en Inglés | MEDLINE | ID: mdl-29693588

RESUMEN

The complement system is a part of the innate immune system that viruses need to face during infections. Many viruses incorporate cellular regulators of complement activation (RCA) to block complement pathways and our prior work has shown that Parainfluenza virus 5 (PIV5) incorporates CD55 and CD46 to delay complement-mediated neutralization. In this paper, we tested the role of a third individual RCA inhibitor CD59 in PIV5 interactions with complement pathways. Using a cell line engineered to express CD59, we show that small levels of functional CD59 are associated with progeny PIV5, which is capable of blocking assembly of the C5b-C9 membrane attack complex (MAC). PIV5 containing CD59 (PIV5-CD59) showed increased resistance to complement-mediated neutralization in vitro comparing to PIV5 lacking regulators. Infection of A549 cells with PIV5 and RSV upregulated CD59 expression. TGF-beta treatment of PIV5-infected cells also increased cell surface CD59 expression and progeny virions were more resistant to complement-mediated neutralization. A comparison of individual viruses containing only CD55, CD46, or CD59 showed a potency of inhibiting complement-mediated neutralization, which followed a pattern of CD55 > CD46 > CD59.


Asunto(s)
Antígenos CD55/metabolismo , Antígenos CD59/metabolismo , Activación de Complemento/inmunología , Complemento C9/metabolismo , Inactivadores del Complemento/metabolismo , Proteína Cofactora de Membrana/metabolismo , Virus de la Parainfluenza 5/inmunología , Animales , Antígenos CD59/genética , Bovinos , Línea Celular , Membrana Celular/metabolismo , Chlorocebus aethiops , Cricetinae , Expresión Génica , Interacciones Huésped-Patógeno/inmunología , Humanos , Pruebas de Neutralización , Virus de la Parainfluenza 5/metabolismo , Virión/metabolismo
16.
Virulence ; 9(1): 70-82, 2018 01 01.
Artículo en Inglés | MEDLINE | ID: mdl-28277903

RESUMEN

Staphylococcus aureus is a serious public health burden causing a wide variety of infections. Earlier detection of such infections could result in faster and more directed therapies that also prevent resistance development. Human monoclonal antibodies (humAbs) are promising tools for diagnosis and therapy owing to their relatively straightforward synthesis, long history of safe clinical use and high target specificity. Here we show that the humAb 6D4, which was obtained from a random screen of B-cells producing antibodies that bind to whole cells of S. aureus, targets the staphylococcal complement inhibitor (SCIN). The epitope recognized by 6D4 was localized to residues 26 to 36 in the N-terminus of SCIN, which overlap with the active site. Accordingly, 6D4 can inhibit SCIN activity as demonstrated through the analysis of C3b deposition on S. aureus cells and complement-induced lysis of rabbit erythrocytes. Importantly, while SCIN is generally regarded as a secreted virulence factor, 6D4 allowed detection of strongly increased SCIN binding to S. aureus cells upon exposure to human serum, relating to the known binding of SCIN to C3 convertases deposited on the staphylococcal cell surface. Lastly, we show that labeling of humAb 6D4 with a near-infrared fluorophore allows one-step detection of SCIN-producing S. aureus cells. Together, our findings show that the newly described humAb 6D4 specifically recognizes S. aureus SCIN, which can potentially be used for detection of human serum-incubated S. aureus strains expressing SCIN.


Asunto(s)
Anticuerpos Antibacterianos/metabolismo , Anticuerpos Monoclonales/metabolismo , Antígenos Bacterianos/metabolismo , Inactivadores del Complemento/metabolismo , Infecciones Estafilocócicas/inmunología , Staphylococcus aureus/patogenicidad , Factores de Virulencia/metabolismo , Animales , Anticuerpos Antibacterianos/química , Anticuerpos Monoclonales/química , Antígenos Bacterianos/química , Dominio Catalítico , Convertasas de Complemento C3-C5/metabolismo , Complemento C3b/metabolismo , Inactivadores del Complemento/química , Colorantes Fluorescentes/química , Humanos , Microscopía Fluorescente , Unión Proteica , Conejos , Factores de Virulencia/química
17.
Mol Immunol ; 89: 111-114, 2017 09.
Artículo en Inglés | MEDLINE | ID: mdl-28610663

RESUMEN

The complement system has obtained renewed clinical focus due to increasing number of patients treated with eculizumab, a monoclonal antibody inhibiting cleavage of C5 into C5a and C5b. The FDA approved indications are paroxysmal nocturnal haemoglobinuria and atypical haemolytic uremic syndrome, but many other diseases are candidates for complement inhibition. It has been postulated that eculizumab does not inhibit C5a formation in vivo, in contrast to what would be expected since it blocks C5 cleavage. We recently revealed that this finding was due to a false positive reaction in a C5a assay. In the present study, we identified expression of a neoepitope which was exposed on C5 after binding to eculizumab in vivo. By size exclusion chromatography of patient serum obtained before and after infusion of eculizumab, we document that the neoepitope was exposed in the fractions containing the eculizumab-C5 complexes, being positive in this actual C5a assay and negative in others. Furthermore, we confirmed that it was the eculizumab-C5 complexes that were detected in the C5a assay by adding an anti-IgG4 antibody as detection antibody. Competitive inhibition by anti-C5 antibodies localized the epitope to the C5a moiety of C5. Finally, acidification of C5, known to alter C5 conformation, induced a neoepitope reacting identical to the one we explored, in the C5a assays. These data are important for interpretation of complement analyses in patients treated with eculizumab.


Asunto(s)
Anticuerpos Monoclonales Humanizados/uso terapéutico , Activación de Complemento/efectos de los fármacos , Complemento C5/inmunología , Complemento C5a/inmunología , Complemento C5b/inmunología , Anticuerpos Monoclonales Humanizados/metabolismo , Síndrome Hemolítico Urémico Atípico/sangre , Síndrome Hemolítico Urémico Atípico/tratamiento farmacológico , Síndrome Hemolítico Urémico Atípico/inmunología , Cromatografía en Gel , Activación de Complemento/inmunología , Complemento C5/metabolismo , Complemento C5a/metabolismo , Complemento C5b/metabolismo , Inactivadores del Complemento/metabolismo , Inactivadores del Complemento/uso terapéutico , Ensayo de Inmunoadsorción Enzimática , Epítopos/inmunología , Epítopos/metabolismo , Hemoglobinuria Paroxística/sangre , Hemoglobinuria Paroxística/tratamiento farmacológico , Hemoglobinuria Paroxística/inmunología , Humanos , Concentración de Iones de Hidrógeno , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Evaluación de Resultado en la Atención de Salud , Unión Proteica/inmunología
18.
PLoS Negl Trop Dis ; 11(3): e0005437, 2017 03.
Artículo en Inglés | MEDLINE | ID: mdl-28278252

RESUMEN

BACKGROUND: On a global scale scabies is one of the most common dermatological conditions, imposing a considerable economic burden on individuals, communities and health systems. There is substantial epidemiological evidence that in tropical regions scabies is often causing pyoderma and subsequently serious illness due to invasion by opportunistic bacteria. The health burden due to complicated scabies causing cellulitis, bacteraemia and sepsis, heart and kidney diseases in resource-poor communities is extreme. Co-infections of group A streptococcus (GAS) and scabies mites is a common phenomenon in the tropics. Both pathogens produce multiple complement inhibitors to overcome the host innate defence. We investigated the relative role of classical (CP), lectin (LP) and alternative pathways (AP) towards a pyodermic GAS isolate 88/30 in the presence of a scabies mite complement inhibitor, SMSB4. METHODOLOGY/PRINCIPAL FINDINGS: Opsonophagocytosis assays in fresh blood showed baseline immunity towards GAS. The role of innate immunity was investigated by deposition of the first complement components of each pathway, specifically C1q, FB and MBL from normal human serum on GAS. C1q deposition was the highest followed by FB deposition while MBL deposition was undetectable, suggesting that CP and AP may be mainly activated by GAS. We confirmed this result using sera depleted of either C1q or FB, and serum deficient in MBL. Recombinant SMSB4 was produced and purified from Pichia pastoris. SMSB4 reduced the baseline immunity against GAS by decreasing the formation of CP- and AP-C3 convertases, subsequently affecting opsonisation and the release of anaphylatoxin. CONCLUSIONS/SIGNIFICANCE: Our results indicate that the complement-inhibitory function of SMSB4 promotes the survival of GAS in vitro and inferably in the microenvironment of the mite-infested skin. Understanding the tripartite interactions between host, parasite and microbial pathogens at a molecular level may serve as a basis to develop improved intervention strategies targeting scabies and associated bacterial infections.


Asunto(s)
Inactivadores del Complemento/metabolismo , Proteínas del Sistema Complemento/inmunología , Factores Inmunológicos/antagonistas & inhibidores , Sarcoptes scabiei/metabolismo , Streptococcus pyogenes/crecimiento & desarrollo , Streptococcus pyogenes/inmunología , Animales , Humanos , Viabilidad Microbiana , Proteínas Opsoninas/metabolismo , Piodermia/etiología , Escabiosis/complicaciones
19.
J Immunol ; 198(6): 2330-2340, 2017 03 15.
Artículo en Inglés | MEDLINE | ID: mdl-28148731

RESUMEN

Respiratory tract infections are one of the leading causes of mortality worldwide urging better understanding of interactions between pathogens causing these infections and the host. Here we report that an extracellular matrix component proline/arginine-rich end leucine-rich repeat protein (PRELP) is a novel antibacterial component of innate immunity. We detected the presence of PRELP in human bronchoalveolar lavage fluid and showed that PRELP can be found in alveolar fluid, resident macrophages/monocytes, myofibroblasts, and the adventitia of blood vessels in lung tissue. PRELP specifically binds respiratory tract pathogens Moraxella catarrhalis, Haemophilus influenzae, and Streptococcus pneumoniae, but not other bacterial pathogens tested. We focused our study on M. catarrhalis and found that PRELP binds the majority of clinical isolates of M. catarrhalis (n = 49) through interaction with the ubiquitous surface protein A2/A2H. M. catarrhalis usually resists complement-mediated serum killing by recruiting to its surface a complement inhibitor C4b-binding protein, which is also a ligand for PRELP. We found that PRELP competitively inhibits binding of C4b-binding protein to bacteria, which enhances membrane attack complex formation on M. catarrhalis and thus leads to increased serum sensitivity. Furthermore, PRELP enhances phagocytic killing of serum-opsonized M. catarrhalis by human neutrophils in vitro. Moreover, PRELP reduces Moraxella adherence to and invasion of human lung epithelial A549 cells. Taken together, PRELP enhances host innate immunity against M. catarrhalis through increasing complement-mediated attack, improving phagocytic killing activity of neutrophils, and preventing bacterial adherence to lung epithelial cells.


Asunto(s)
Proteínas de la Matriz Extracelular/metabolismo , Glicoproteínas/metabolismo , Macrófagos/inmunología , Moraxella catarrhalis/inmunología , Infecciones por Moraxellaceae/inmunología , Miofibroblastos/inmunología , Mucosa Respiratoria/inmunología , Infecciones del Sistema Respiratorio/inmunología , Citotoxicidad Celular Dependiente de Anticuerpos , Adhesión Bacteriana , Línea Celular , Inactivadores del Complemento/antagonistas & inhibidores , Inactivadores del Complemento/metabolismo , Interacciones Huésped-Patógeno , Humanos , Evasión Inmune , Inmunidad Innata , Fagocitosis , Mucosa Respiratoria/patología
20.
Exp Biol Med (Maywood) ; 242(4): 397-410, 2017 02.
Artículo en Inglés | MEDLINE | ID: mdl-27798122

RESUMEN

The complement system is one of the body's major innate immune defense mechanisms in vertebrates. Its function is to detect foreign bodies and promote their elimination through opsonisation or lysis. Complement proteins play an important role in the immunopathogenesis of several disorders. However, excessive complement activation does not confer more protection but instead leads to several autoimmune and inflammatory diseases. With inappropriate activation of the complement system, activated complement proteins and glycoproteins may damage both healthy and diseased tissues. Development of complement inhibitors represents an effective approach in controlling dysregulated complement activity and reducing disease severity, yet few studies have investigated the nature and role of novel complement inhibitory proteins of viral origin. Viral complement inhibitors have important implications in understanding the importance of complement inhibition and their role as a promising novel therapeutic approach in diseases caused by dysregulated complement function. In this review, we discuss the role and importance of complement inhibitors derived from several viruses in the scope of human inflammatory and autoimmune diseases.


Asunto(s)
Activación de Complemento/inmunología , Inactivadores del Complemento/metabolismo , Proteínas Virales/metabolismo , Virus/inmunología , Animales , Enfermedades Autoinmunes/inmunología , Proteínas del Sistema Complemento/metabolismo , Humanos , Inmunomodulación/inmunología , Inflamación/inmunología , Ratones , Proteínas Virales/farmacología , Virosis/inmunología
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