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1.
Kidney Int ; 94(4): 689-700, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-29884545

RESUMO

Certain kidney diseases are associated with complement activation although a renal triggering factor has not been identified. Here we demonstrated that renin, a kidney-specific enzyme, cleaves C3 into C3b and C3a, in a manner identical to the C3 convertase. Cleavage was specifically blocked by the renin inhibitor aliskiren. Renin-mediated C3 cleavage and its inhibition by aliskiren also occurred in serum. Generation of C3 cleavage products was demonstrated by immunoblotting, detecting the cleavage product C3b, by N-terminal sequencing of the cleavage product, and by ELISA for C3a release. Functional assays showed mast cell chemotaxis towards the cleavage product C3a and release of factor Ba when the cleavage product C3b was combined with factor B and factor D. The renin-mediated C3 cleavage product bound to factor B. In the presence of aliskiren this did not occur, and less C3 deposited on renin-producing cells. The effect of aliskiren was studied in three patients with dense deposit disease and this demonstrated decreased systemic and renal complement activation (increased C3, decreased C3a and C5a, decreased renal C3 and C5b-9 deposition and/or decreased glomerular basement membrane thickness) over a follow-up period of four to seven years. Thus, renin can trigger complement activation, an effect inhibited by aliskiren. Since renin concentrations are higher in renal tissue than systemically, this may explain the renal propensity of complement-mediated disease in the presence of complement mutations or auto-antibodies.


Assuntos
Amidas/farmacologia , Ativação do Complemento/efeitos dos fármacos , Complemento C3/química , Fumaratos/farmacologia , Glomerulonefrite Membranoproliferativa/metabolismo , Glomerulonefrite Membranoproliferativa/terapia , Renina/química , Amidas/uso terapêutico , Quimiotaxia/efeitos dos fármacos , Criança , Complemento C3/metabolismo , Complemento C3a/química , Complemento C3a/metabolismo , Complemento C3b/química , Complemento C3b/metabolismo , Complemento C4/química , Complemento C5a/química , Complemento C5a/metabolismo , Complemento C5b/química , Complemento C5b/metabolismo , Fator B do Complemento/química , Fator D do Complemento/química , Feminino , Fumaratos/uso terapêutico , Membrana Basal Glomerular/patologia , Glomerulonefrite Membranoproliferativa/patologia , Humanos , Mastócitos/fisiologia , Renina/antagonistas & inibidores , Renina/metabolismo
2.
J Immunotoxicol ; 15(1): 63-72, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-29534626

RESUMO

Both NF-κB pathway and complement activation appear to be involved in kidney damage induced by trichloroethylene (TCE). However, any relationship between these two systems has not yet been established. The present study aimed to clarify the role of NF-κB in complement activation and renal injury in TCE-sensitized BALB/c mice. Mice were sensitized by an initial subcutaneous injection and repeated focal applications of TCE to dorsal skin at specified timepoints. NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC) was injected (intraperitoneal) before the final two focal TCE challenges. In the experiments, mice had their blood and kidneys collected. Kidney function was evaluated via blood urea nitrogen (BUN) and creatinine (Cr) content; renal histology was examined using transmission electron microscopy (TEM). Kidney levels of phospho-p65 were assessed by Western blot and kidney mRNA levels of interleukin (IL)-1ß, IL-6, IL-17, tumor necrosis factor (TNF)-α, and p65 by real-time quantitative PCR. Presence of C3 and C5b-9 membrane attack complexes in the kidneys was evaluated via immunohistochemistry. The results showed there was significant swelling, vacuolar degeneration in mitochondria, shrinkage of microvilli, disappearance of brush borders, segmental foot process fusion, and glomerular basement membrane thickening (or disrobing) in kidneys from TCE-sensitized mice. In conjunction with these changes, serum BUN and Cr levels were increased and IL-1ß, IL-6, IL-17, and TNFα mRNA levels were elevated. Levels of p65 and phospho-p65 protein were also up-regulated, and there was significant C3 and C5b-9 deposition. PDTC pretreatment attenuated TCE-induced up-regulation of p65 and its phosphorylation, complement deposition, cytokine release, and renal damage. These results provide the first evidence that NF-κB pathway has an important role in TCE-induced renal damage mediated by enhanced complement activation in situ.


Assuntos
Lesão Renal Aguda/imunologia , Rim/fisiologia , NF-kappa B/metabolismo , Lesão Renal Aguda/induzido quimicamente , Animais , Nitrogênio da Ureia Sanguínea , Complemento C3/metabolismo , Complemento C5b/metabolismo , Creatinina/sangue , Citocinas/genética , Citocinas/metabolismo , Regulação da Expressão Gênica , Rim/patologia , Camundongos , Camundongos Endogâmicos BALB C , NF-kappa B/antagonistas & inibidores , Pirrolidinas/administração & dosagem , Pirrolidinas/farmacologia , Transdução de Sinais , Tiocarbamatos/administração & dosagem , Tiocarbamatos/farmacologia , Tricloroetileno/toxicidade
3.
Br J Dermatol ; 179(2): 413-419, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29405257

RESUMO

BACKGROUND: Despite the heavy purulence observed in hidradenitis suppurativa (HS), the kinetics of complement anaphylatoxins acting to prime chemotaxis of neutrophils has not been studied. OBJECTIVES: To explore complement activation in HS. METHODS: Circulating concentrations of complement factor C5a, as well as of membrane attack complex C5b-9, were determined in the plasma of 54 treatment-naïve patients and of 14 healthy controls, as well as in the pus of seven patients. Results were correlated with Hurley stage and International Hidradenitis Suppurativa Severity Score. Peripheral blood mononuclear cells (PBMCs) were isolated from seven patients with Hurley stage III HS and seven healthy volunteers and stimulated in the presence of 25% of plasma for the production of tumour necrosis factor-α (TNF-α). RESULTS: Circulating C5a and C5b-9 were significantly greater in patient than in control plasma; however, concentrations in pus were very low. Circulating C5a levels exceeding 28 ng mL-1 were associated with a specificity > 90% with the occurrence of HS. Circulating levels of C5a and C5b-9 were greater in patients with more severe HS. PBMCs of patients produced high concentrations of TNF-α only when growth medium was enriched with patient plasma; this was reversed with the addition of the C5a blocker IFX-1. CONCLUSIONS: Systemic complement activation occurs in HS and may be used as a surrogate biomarker of HS. C5a stimulates overproduction of TNF-α and may be a future therapeutic target.


Assuntos
Ativação do Complemento/imunologia , Complemento C5a/análise , Complemento C5b/análise , Hidradenite Supurativa/imunologia , Adulto , Biomarcadores/sangue , Quimiotaxia de Leucócito/imunologia , Complemento C5a/imunologia , Complemento C5b/imunologia , Feminino , Hidradenite Supurativa/sangue , Hidradenite Supurativa/diagnóstico , Humanos , Masculino , Pessoa de Meia-Idade , Neutrófilos/imunologia , Neutrófilos/metabolismo , Índice de Gravidade de Doença , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismo
4.
Int J Nanomedicine ; 12: 3927-3940, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28579778

RESUMO

Iron oxide nanoparticles (IONPs) are promising nanomaterials for biomedical applications. However, their inflammatory potential has not been fully established. Here, we used a lepirudin anti-coagulated human whole blood model to evaluate the potential of 10 nm IONPs to activate the complement system and induce cytokine production. Reactive oxygen species and cell death were also assessed. The IONPs activated complement, as measured by C3a, C5a and sC5b-9, and induced the production of pro-inflammatory cytokines in a particle-dose dependent manner, with the strongest response at 10 µg/mL IONPs. Complement inhibitors at C3 (compstatin analog Cp40) and C5 (eculizumab) levels completely inhibited complement activation and secretion of inflammatory mediators induced by the IONPs. Additionally, blockade of complement receptors C3aR and C5aR1 significantly reduced the levels of various cytokines, indicating that the particle-induced secretion of inflammatory mediators is mainly C5a and C3a mediated. The IONPs did not induce cell death or reactive oxygen species, which further suggests that complement activation alone was responsible for most of the particle-induced cytokines. These data suggest that the lepirudin anti-coagulated human whole blood model is a valuable ex vivo system to study the inflammatory potential of IONPs. We conclude that IONPs induce complement-mediated cytokine secretion in human whole blood.


Assuntos
Ativação do Complemento , Citocinas/sangue , Compostos Férricos/química , Nanopartículas Metálicas/química , Anti-Inflamatórios/farmacologia , Anticoagulantes/farmacologia , Sobrevivência Celular , Complemento C3a/metabolismo , Complemento C5a/metabolismo , Complemento C5b/metabolismo , Inativadores do Complemento/farmacologia , Hirudinas/farmacologia , Humanos , Tamanho da Partícula , Espécies Reativas de Oxigênio/sangue , Receptores de Complemento/sangue , Proteínas Recombinantes/farmacologia
5.
Mol Immunol ; 89: 111-114, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28610663

RESUMO

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.


Assuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Ativação do Complemento/efeitos dos fármacos , Complemento C5/imunologia , Complemento C5a/imunologia , Complemento C5b/imunologia , Anticorpos Monoclonais Humanizados/metabolismo , Síndrome Hemolítico-Urêmica Atípica/sangue , Síndrome Hemolítico-Urêmica Atípica/tratamento farmacológico , Síndrome Hemolítico-Urêmica Atípica/imunologia , Cromatografia em Gel , Ativação do Complemento/imunologia , Complemento C5/metabolismo , Complemento C5a/metabolismo , Complemento C5b/metabolismo , Inativadores do Complemento/metabolismo , Inativadores do Complemento/uso terapêutico , Ensaio de Imunoadsorção Enzimática , Epitopos/imunologia , Epitopos/metabolismo , Hemoglobinúria Paroxística/sangue , Hemoglobinúria Paroxística/tratamento farmacológico , Hemoglobinúria Paroxística/imunologia , Humanos , Concentração de Íons de Hidrogênio , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Avaliação de Resultados em Cuidados de Saúde , Ligação Proteica/imunologia
6.
Clin Exp Immunol ; 190(1): 110-121, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28640379

RESUMO

One of the most widespread and effective environmental factors is the infection with enteroviruses (EVs) which accelerate ß cell destruction in type 1 diabetes (T1D). This study represented a comparison between diabetic EV+ and EV- children as well as correlation analysis between autoantibodies, T1D markers, cytokines, complement activation products and anti-coxsackievirus (CV) immunoglobulin (Ig)G. EV RNA was detected in Egyptian children with T1D (26·2%) and healthy controls (0%). Detection of anti-CV IgG in T1D-EV+ resulted in 64% positivity. Within T1D-EV+ , previously diagnosed (PD) showed 74 versus 56% in newly diagnosed (ND) children. Comparisons between populations showed increased levels of haemoglobin A1c (HbA1c), C-reactive protein (CRP), nitric oxide (NO), glutamic acid decarboxylase and insulin and islet cell autoantibodies [glutamic acid decarboxylase autoantibodies (GADA), insulin autoantibodies (IAA) and islet cell cytoplasmic autoantibodies (ICA), respectively], interferon (IFN)-γ, tumour necrosis factor (TNF)-α, interleukin (IL)-1ß, IL -10, IL -12, IL -17, C3d and sC5-9 in T1D-EV+ versus T1D-EV- . Conversely, both IL-20 and transforming growth factor (TGF-ß) decreased in T1D-EV+ versus EV- , while IL-4, -6 and -13 did not show any changes. Correlation analysis showed dependency of accelerated autoimmunity and ß cell destruction on increased IFN-γ, IL-12 and IL-17 versus decreased IL-4, -6 and -13. In conclusion, IFN-γ, IL-12 and IL-17 played an essential role in exacerbating EV+ -T1D, while C3d, sC5b -9, IL-10 and -20 displayed distinct patterns.


Assuntos
Citocinas/metabolismo , Diabetes Mellitus Tipo 1/imunologia , Infecções por Enterovirus/imunologia , Enterovirus/imunologia , Ilhotas Pancreáticas/imunologia , Adolescente , Anticorpos Antivirais/metabolismo , Apoptose , Autoanticorpos/metabolismo , Criança , Pré-Escolar , Ativação do Complemento , Complemento C3d/metabolismo , Complemento C5b/metabolismo , Citocinas/genética , Diabetes Mellitus Tipo 1/complicações , Egito , Infecções por Enterovirus/complicações , Glutamato Descarboxilase/imunologia , Humanos , Insulina/imunologia , Ilhotas Pancreáticas/patologia
7.
J Pediatr Hematol Oncol ; 39(4): 282-286, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28060108

RESUMO

Thrombotic complications are a significant source of morbidity and mortality following hematopoietic stem cell transplants. Among them, transplant-associated thrombotic microangiopathy (TA-TMA) is a well-recognized syndrome that can affect various organ systems. Its etiology is related to endothelial injury accompanied by complement activation. As many of the signs and symptoms of the disease are also encountered in other complications following hematopoietic stem cell transplant, it can often be difficult to establish the diagnosis based on clinical data alone. Histopathologic examination of various tissues may be performed in difficult cases. However, the microscopic features of TA-TMA also overlap with those seen in other posttransplant complications, suggesting a need for additional tests to help in diagnosis. Here we describe a patient who presented with hemolytic anemia, thrombocytopenia, renal and neurological impairment, who also developed significant bloody diarrhea. Flexible sigmoidoscopy with biopsies was performed to determine the exact etiology of his gastrointestinal bleed. A diagnosis of intestinal TA-TMA was established with the use of immunohistochemical stains for complement components C5b-9 and C4d. This is the first report that highlights the utility of complement staining on histologic sections from digestive samples to render a definitive diagnosis of intestinal TA-TMA.


Assuntos
Enteropatias/diagnóstico , Transplante de Células-Tronco/efeitos adversos , Microangiopatias Trombóticas/diagnóstico , Criança , Complemento C4b/análise , Complemento C5b/análise , Hemorragia Gastrointestinal/etiologia , Humanos , Imuno-Histoquímica , Masculino , Fragmentos de Peptídeos/análise , Microangiopatias Trombóticas/patologia
8.
Rheumatology (Oxford) ; 56(1): 77-86, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-28028157

RESUMO

OBJECTIVES: Neuropsychiatric (NP) involvement is a poorly understood manifestation of SLE. We studied post-mortem histopathology in relation to clinical NPSLE syndromes and complement deposition in brains of NPSLE and SLE patients and controls. Furthermore, we investigated the correlation between cerebral post-mortem histopathology and ex vivo 7 T MRI findings in SLE and NPSLE. METHODS: A nationwide search for autopsy material yielded brain tissue from 16 NPSLE and 18 SLE patients. Brains obtained from 24 patients who died of acute cardiac events served as controls. Apart from a histopathological evaluation, paraffin-embedded cortical tissue was stained for components of the classical, lectin and terminal complement pathways. RESULTS: Diffuse vasculopathy, microinfarction, macroinfarction, vasculitis and microthrombi occurred significantly more often in NPSLE than SLE patients and were absent in controls. Focal vasculopathy was found in both SLE patients and controls. Complement deposition was strongly associated with both SLE and NPSLE, but not with controls (P < 0.001). Microthrombi were found uniquely in NPSLE and were associated with C4d and C5b-9 deposits (P < 0.05). A 7 T MRI was unable to detect most small vessel injury that was visible histopathologically. CONCLUSION: Our study demonstrates that histopathological lesions in NPSLE represent a continuum, ranging from non-specific lesions such as focal vasculopathy, to more specific lesions including C4d- and C5b-9-associated microthrombi and diffuse vasculopathy related to clinical syndromes defining NPSLE. Complement deposition may be a key factor in the interaction between circulating autoantibodies and thromboischaemic lesions observed in NPSLE. Therefore, complement inhibition may have novel therapeutic potential in NPSLE.


Assuntos
Infarto Encefálico/patologia , Encéfalo/patologia , Trombose Intracraniana/patologia , Vasculite Associada ao Lúpus do Sistema Nervoso Central/patologia , Vasculite do Sistema Nervoso Central/patologia , Adulto , Idoso , Autoanticorpos/imunologia , Autopsia , Encéfalo/diagnóstico por imagem , Encéfalo/metabolismo , Infarto Encefálico/diagnóstico por imagem , Infarto Encefálico/etiologia , Infarto Encefálico/metabolismo , Estudos de Casos e Controles , Complemento C4b/imunologia , Complemento C4b/metabolismo , Complemento C5b/imunologia , Complemento C5b/metabolismo , Complexo de Ataque à Membrana do Sistema Complemento/imunologia , Complexo de Ataque à Membrana do Sistema Complemento/metabolismo , Via Clássica do Complemento , Lectina de Ligação a Manose da Via do Complemento , Proteínas do Sistema Complemento/imunologia , Proteínas do Sistema Complemento/metabolismo , Feminino , Humanos , Imuno-Histoquímica , Trombose Intracraniana/diagnóstico por imagem , Trombose Intracraniana/etiologia , Trombose Intracraniana/metabolismo , Lúpus Eritematoso Sistêmico/metabolismo , Lúpus Eritematoso Sistêmico/patologia , Vasculite Associada ao Lúpus do Sistema Nervoso Central/complicações , Vasculite Associada ao Lúpus do Sistema Nervoso Central/diagnóstico por imagem , Vasculite Associada ao Lúpus do Sistema Nervoso Central/metabolismo , Imagem por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/metabolismo , Vasculite do Sistema Nervoso Central/diagnóstico por imagem , Vasculite do Sistema Nervoso Central/etiologia , Vasculite do Sistema Nervoso Central/metabolismo
9.
Sci Rep ; 6: 30239, 2016 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-27444648

RESUMO

Terminal complement membrane attack complex (MAC) formation is induced initially by C5b, followed by the sequential condensation of the C6, C7, C8. Polymerization of C9 to the C5b-8 complex forms the C5b-9 (or MAC). The C5b-9 forms lytic or non lytic pores in the cell membrane destroys membrane integrity. The biological functionalities of MAC has been previously investigated by using either the mice deficient in C5 and C6, or MAC's regulator CD59. However, there is no available C9 deficient mice (mC9(-/-)) for directly dissecting the role of C5b-9 in the pathogenesis of human diseases. Further, since C5b-7 and C5b-8 complexes form non lytic pore, it may also plays biological functionality. To better understand the role of terminal complement cascades, here we report a successful generation of mC9(-/-). We demonstrated that lack of C9 attenuates anti-erythrocyte antibody-mediated hemolysis or LPS-induced acute shock. Further, the rescuing effect on the acute shock correlates with the less release of IL-1ß in mC9(-/-), which is associated with suppression of MAC-mediated inflammasome activation in mC9(-/-). Taken together, these results not only confirm the critical role of C5b-9 in complement-mediated hemolysis and but also highlight the critical role of C5b-9 in inflammasome activation.


Assuntos
Complemento C5b/genética , Complemento C9/genética , Complexo de Ataque à Membrana do Sistema Complemento/genética , Inflamação/genética , Choque/genética , Animais , Anticorpos/imunologia , Anticorpos/metabolismo , Membrana Celular/genética , Membrana Celular/metabolismo , Complemento C5b/imunologia , Complemento C9/imunologia , Complexo de Ataque à Membrana do Sistema Complemento/química , Complexo de Ataque à Membrana do Sistema Complemento/imunologia , Proteínas do Sistema Complemento/genética , Proteínas do Sistema Complemento/imunologia , Proteínas do Sistema Complemento/metabolismo , Eritrócitos/imunologia , Eritrócitos/metabolismo , Hemólise/imunologia , Humanos , Inflamassomos/genética , Inflamassomos/imunologia , Inflamação/induzido quimicamente , Inflamação/imunologia , Inflamação/patologia , Lipopolissacarídeos/toxicidade , Camundongos , Camundongos Knockout , Choque/induzido quimicamente , Choque/imunologia , Choque/fisiopatologia
10.
J Immunotoxicol ; 13(4): 567-79, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27027470

RESUMO

Trichloroethylene (TCE) is a major environmental pollutant. An immunological response is a newly-recognized mechanism for TCE-induced kidney damage. However, the role of the plasma kallikrein-kinin system (KKS) in immune-mediated kidney injury has never been examined. This study aimed to explore the role of the key components of the KKS, i.e. plasma kallikrein (PK), bradykinin (BK) and its receptors B1R and B2R, in TCE-induced kidney injury. A mouse model of skin sensitization was used to explore the mechanism of injury with or without a PK inhibitor PKSI. Kidney function was evaluated by measuring blood urea nitrogen (BUN) and creatinine (Cr) in conjunction with histopathologic characterization. Plasma BK was determined by ELISA; Renal C5b-9 membrane attack complex was evaluated by immunohistochemistry. Expression of BK and PK in the kidney was detected by immunofluorescence. mRNA and protein levels of B1R and B2R were assessed by real-time qPCR and Western blot. As expected, numerous inflammatory cell infiltration and tubular epithelial cell vacuolar degeneration were observed in TCE-sensitized mice. Moreover, serum BUN and Cr and plasma BK were increased. In addition, deposition of BK, PK and C5b-9 were observed and B1R and B2R mRNA and proteins levels were up-regulated. Pre-treatment with PKSI, a highly selective inhibitor of PK, alleviated TCE-induced renal damage. In addition, PKSI attenuated TCE-induced up-regulation of BK, PK and its receptors and C5b-9. These results provided the first evidence that activation of the KKS contributed to immune-mediated renal injury induced by TCE and also helped to identify the KKS as a potential therapeutic target for mitigating chemical sensitization-induced renal damage.


Assuntos
Lesão Renal Aguda/imunologia , Poluição Ambiental/efeitos adversos , Sistema Calicreína-Cinina , Tricloroetileno/toxicidade , Urotélio/patologia , Animais , Nitrogênio da Ureia Sanguínea , Bradicinina/sangue , Complemento C5b/metabolismo , Creatinina/sangue , Feminino , Regulação da Expressão Gênica , Humanos , Calicreínas/sangue , Camundongos , Camundongos Endogâmicos BALB C , Receptor B1 da Bradicinina/genética , Receptor B1 da Bradicinina/metabolismo , Receptor B2 da Bradicinina/genética , Receptor B2 da Bradicinina/metabolismo
11.
Nat Commun ; 7: 10587, 2016 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-26841837

RESUMO

In response to complement activation, the membrane attack complex (MAC) assembles from fluid-phase proteins to form pores in lipid bilayers. MAC directly lyses pathogens by a 'multi-hit' mechanism; however, sublytic MAC pores on host cells activate signalling pathways. Previous studies have described the structures of individual MAC components and subcomplexes; however, the molecular details of its assembly and mechanism of action remain unresolved. Here we report the electron cryo-microscopy structure of human MAC at subnanometre resolution. Structural analyses define the stoichiometry of the complete pore and identify a network of interaction interfaces that determine its assembly mechanism. MAC adopts a 'split-washer' configuration, in contrast to the predicted closed ring observed for perforin and cholesterol-dependent cytolysins. Assembly precursors partially penetrate the lipid bilayer, resulting in an irregular ß-barrel pore. Our results demonstrate how differences in symmetric and asymmetric components of the MAC underpin a molecular basis for pore formation and suggest a mechanism of action that extends beyond membrane penetration.


Assuntos
Complemento C5b/ultraestrutura , Complemento C6/ultraestrutura , Complemento C7/ultraestrutura , Complemento C8/ultraestrutura , Complemento C9/ultraestrutura , Complexo de Ataque à Membrana do Sistema Complemento/ultraestrutura , Complexos Multiproteicos/ultraestrutura , Cromatografia Líquida , Microscopia Crioeletrônica , Corantes Fluorescentes , Humanos , Processamento de Imagem Assistida por Computador , Espectrometria de Massas , Microscopia Eletrônica , Modelos Moleculares , Estrutura Molecular , Estrutura Secundária de Proteína
12.
PLoS One ; 10(9): e0136558, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26335102

RESUMO

BACKGROUND: Recent pre-clinical studies have shown that complement activation contributes to glomerular and tubular injury in experimental FSGS. Although complement proteins are detected in the glomeruli of some patients with FSGS, it is not known whether this is due to complement activation or whether the proteins are simply trapped in sclerotic glomeruli. We measured complement activation fragments in the plasma and urine of patients with primary FSGS to determine whether complement activation is part of the disease process. STUDY DESIGN: Plasma and urine samples from patients with biopsy-proven FSGS who participated in the FSGS Clinical Trial were analyzed. SETTING AND PARTICIPANTS: We identified 19 patients for whom samples were available from weeks 0, 26, 52 and 78. The results for these FSGS patients were compared to results in samples from 10 healthy controls, 10 patients with chronic kidney disease (CKD), 20 patients with vasculitis, and 23 patients with lupus nephritis. OUTCOMES: Longitudinal control of proteinuria and estimated glomerular filtration rate (eGFR). MEASUREMENTS: Levels of the complement fragments Ba, Bb, C4a, and sC5b-9 in plasma and urine. RESULTS: Plasma and urine Ba, C4a, sC5b-9 were significantly higher in FSGS patients at the time of diagnosis than in the control groups. Plasma Ba levels inversely correlated with the eGFR at the time of diagnosis and at the end of the study. Plasma and urine Ba levels at the end of the study positively correlated with the level of proteinuria, the primary outcome of the study. LIMITATIONS: Limited number of patients with samples from all time-points. CONCLUSIONS: The complement system is activated in patients with primary FSGS, and elevated levels of plasma Ba correlate with more severe disease. Measurement of complement fragments may identify a subset of patients in whom the complement system is activated. Further investigations are needed to confirm our findings and to determine the prognostic significance of complement activation in patients with FSGS.


Assuntos
Ativação do Complemento , Glomerulosclerose Segmentar e Focal/sangue , Glomerulosclerose Segmentar e Focal/urina , Adolescente , Adulto , Criança , Complemento C5b/metabolismo , Complemento C5b/urina , Feminino , Humanos , Masculino
13.
Apoptosis ; 20(4): 433-43, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25735751

RESUMO

The loss of photoreceptors is the defining characteristic of many retinal degenerative diseases, but the mechanisms that regulate photoreceptor cell death are not fully understood. Here we have used the 661W cone photoreceptor cell line to ask whether exposure to the terminal complement complex C5b-9 induces cell death and/or modulates the sensitivity of these cells to other cellular stressors. 661W cone photoreceptors were exposed to complete normal human serum following antibody blockade of CD59. Apoptosis induction was assessed morphologically, by flow cytometry, and on western blotting by probing for cleaved PARP and activated caspase-3. Necroptosis was assessed by flow cytometry and Sirtuin 2 inhibition using 2-cyano-3-[5-(2,5-dichlorophenyl)-2-furyl]-N-5-quinolinylacrylamide (AGK2). The sensitivity of 661W cells to ionomycin, staurosporine, peroxide and chelerythrine was also investigated, with or without prior formation of C5b-9. 661W cells underwent apoptotic cell death following exposure to C5b-9, as judged by poly(ADP-ribose) polymerase 1 cleavage and activation of caspase-3. We also observed apoptotic cell death in response to staurosporine, but 661W cells were resistant to both ionomycin and peroxide. Interestingly, C5b-9 significantly increased 661W sensitivity to staurosporine-induced apoptosis and necroptosis. These studies show that low levels of C5b-9 on 661W cells can induce apoptosis, and that C5b-9 specifically sensitizes 661W cells to certain apoptotic and necroptotic pathways. Our observations provide new insight into the potential role of the complement system in photoreceptor loss, with implications for the molecular aetiology of retinal disease.


Assuntos
Apoptose , Complemento C5b/metabolismo , Complemento C6/metabolismo , Complemento C7/metabolismo , Complemento C8/metabolismo , Complemento C9/metabolismo , Células Fotorreceptoras/citologia , Células Fotorreceptoras/metabolismo , Caspase 3/metabolismo , Linhagem Celular , Complexo de Ataque à Membrana do Sistema Complemento/metabolismo , Humanos , Necrose
14.
Mol Immunol ; 66(2): 164-70, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25795308

RESUMO

An emerging number of diseases and therapeutic approaches with defined involvement of the complement system justify a need for specific markers reflecting activation of particular effector arms of the complement cascade. Measurement of such soluble markers in circulation is a challenge since the specificity of antibodies must be limited to activated complement fragments but not predominant and ubiquitous parental molecules. Existing assays for the measurement of soluble, activated complement proteins are based on the detection of conformational neoepitopes. We tested an alternative approach based on detection of short linear neoepitopes exposed at the cleavage sites after activation of the actual complement component. Obtained antibodies reactive to C4d and C5b fragments enabled us to set up highly specific sandwich ELISAs, which ensured trustful measurements without false positive readouts characteristic for some of the widely used assays.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica , Complemento C5b/metabolismo , Ensaio de Imunoadsorção Enzimática/métodos , Neoplasias Hematológicas/sangue , Fragmentos de Peptídeos/sangue , Animais , Anticorpos/química , Anticorpos/isolamento & purificação , Ativação do Complemento , Complemento C4b/química , Complemento C4b/imunologia , Complemento C5b/química , Complemento C5b/imunologia , Neoplasias Hematológicas/tratamento farmacológico , Neoplasias Hematológicas/imunologia , Neoplasias Hematológicas/patologia , Humanos , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/imunologia , Proteólise , Coelhos , Sensibilidade e Especificidade
15.
Stem Cell Rev Rep ; 11(1): 110-8, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25086571

RESUMO

Activation of complement cascade (ComC) play and important role in mobilization of hematopoietic stem/progenitor cells (HSPCs) from bone marrow (BM) into peripheral blood (PB). While there are vast experimental data on the mechanisms and factors that induce or promote mobilization of HSPCs, there is relatively less data on negative regulators of this process. We demonstrate for the first time that heme oxygenase-1 (HO-1) that has a well-documented anti-inflammatory potential plays an important and heretofore unrecognized role in retention of HSPCs in BM niches by i) modulating negatively activation of mobilization promoting ComC, ii) maintaining stromal derived factor-1 (SDF-1) level in the BM microenvironment and iii) attenuating chemotactic responsiveness of HSPCs to SDF-1 and sphingosine-1 phosphate (S1P) gradients in PB. Furthermore, our data showing a positive mobilizing effect by a non-toxic small-molecule inhibitor of HO-1 (SnPP) suggest that blockade of HO-1 would be a promising strategy to facilitate mobilization of HSPCs. Further studies are also needed to evaluate better the molecular mechanisms responsible for the potential effect of HO-1 in homing of HSPCs after transplantation.


Assuntos
Movimento Celular , Mobilização de Células-Tronco Hematopoéticas/métodos , Células-Tronco Hematopoéticas/metabolismo , Heme Oxigenase-1/metabolismo , Animais , Adesão Celular , Quimiocina CXCL12/metabolismo , Ensaio de Unidades Formadoras de Colônias , Complemento C5b/metabolismo , Complemento C9/metabolismo , Citometria de Fluxo , Fator Estimulador de Colônias de Granulócitos/farmacologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Heme Oxigenase-1/antagonistas & inibidores , Heme Oxigenase-1/genética , Contagem de Leucócitos , Lisofosfolipídeos/metabolismo , Camundongos da Linhagem 129 , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Biológicos , Protoporfirinas/farmacologia , Esfingosina/análogos & derivados , Esfingosina/metabolismo
16.
J Immunol ; 193(10): 5099-107, 2014 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-25297874

RESUMO

Listeria monocytogenes is a major cause of mortality resulting from food poisoning in the United States. In mice, C5 has been genetically linked to host resistance to listeriosis. Despite this genetic association, it remains poorly understood how C5 and its activation products, C5a and C5b, confer host protection to this Gram-positive intracellular bacterium. In this article, we show in a systemic infection model that the major receptor for C5a, C5aR1, is required for a normal robust host immune response against L. monocytogenes. In comparison with wild-type mice, C5aR1(-/-) mice had reduced survival and increased bacterial burden in their livers and spleens. Infected C5aR1(-/-) mice exhibited a dramatic reduction in all major subsets of splenocytes, which was associated with elevated caspase-3 activity and increased TUNEL staining. Because type 1 IFN has been reported to impede the host response to L. monocytogenes through the promotion of splenocyte death, we examined the effect of C5aR1 on type 1 IFN expression in vivo. Indeed, serum levels of IFN-α and IFN-ß were significantly elevated in L. monocytogenes-infected C5aR1(-/-) mice. Similarly, the expression of TRAIL, a type 1 IFN target gene and a proapoptotic factor, was elevated in NK cells isolated from infected C5aR1(-/-) mice. Treatment of C5aR1(-/-) mice with a type 1 IFNR blocking Ab resulted in near-complete rescue of L. monocytogenes-induced mortality. Thus, these findings reveal a critical role for C5aR1 in host defense against L. monocytogenes through the suppression of type 1 IFN expression.


Assuntos
Interferon-alfa/genética , Interferon beta/genética , Listeria monocytogenes/imunologia , Listeriose/imunologia , Baço/imunologia , Anafilatoxinas/imunologia , Animais , Anticorpos/farmacologia , Apoptose , Carga Bacteriana , Caspase 3/genética , Caspase 3/imunologia , Complemento C5a/genética , Complemento C5a/imunologia , Complemento C5b/genética , Complemento C5b/imunologia , Expressão Gênica , Interferon-alfa/imunologia , Interferon beta/imunologia , Listeriose/tratamento farmacológico , Listeriose/microbiologia , Listeriose/mortalidade , Fígado/imunologia , Fígado/microbiologia , Fígado/patologia , Linfócitos/imunologia , Linfócitos/microbiologia , Linfócitos/patologia , Masculino , Camundongos , Camundongos Knockout , Receptor da Anafilatoxina C5a/genética , Receptor da Anafilatoxina C5a/imunologia , Receptores de Interferon/antagonistas & inibidores , Receptores de Interferon/genética , Receptores de Interferon/imunologia , Baço/microbiologia , Baço/patologia , Análise de Sobrevida , Ligante Indutor de Apoptose Relacionado a TNF/genética , Ligante Indutor de Apoptose Relacionado a TNF/imunologia
17.
PLoS Pathog ; 10(9): e1004412, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25254972

RESUMO

During evolution, herpesviruses have developed numerous, and often very ingenious, strategies to counteract efficient host immunity. Specifically, Kaposi's sarcoma-associated herpesvirus (KSHV) eludes host immunity by undergoing a dormant stage, called latency wherein it expresses a minimal number of viral proteins to evade host immune activation. Here, we show that during latency, KSHV hijacks the complement pathway to promote cell survival. We detected strong deposition of complement membrane attack complex C5b-9 and the complement component C3 activated product C3b on Kaposi's sarcoma spindle tumor cells, and on human endothelial cells latently infected by KSHV, TIME-KSHV and TIVE-LTC, but not on their respective uninfected control cells, TIME and TIVE. We further showed that complement activation in latently KSHV-infected cells was mediated by the alternative complement pathway through down-regulation of cell surface complement regulatory proteins CD55 and CD59. Interestingly, complement activation caused minimal cell death but promoted the survival of latently KSHV-infected cells grown in medium depleted of growth factors. We found that complement activation increased STAT3 tyrosine phosphorylation (Y705) of KSHV-infected cells, which was required for the enhanced cell survival. Furthermore, overexpression of either CD55 or CD59 in latently KSHV-infected cells was sufficient to inhibit complement activation, prevent STAT3 Y705 phosphorylation and abolish the enhanced survival of cells cultured in growth factor-depleted condition. Together, these results demonstrate a novel mechanism by which an oncogenic virus subverts and exploits the host innate immune system to promote viral persistent infection.


Assuntos
Apoptose/imunologia , Complemento C3b/metabolismo , Complemento C5b/metabolismo , Herpesvirus Humano 8/fisiologia , Sarcoma de Kaposi/virologia , Latência Viral , Western Blotting , Proliferação de Células , Células Cultivadas , Complemento C3b/genética , Complemento C5b/genética , Endotélio Vascular/imunologia , Endotélio Vascular/patologia , Endotélio Vascular/virologia , Citometria de Fluxo , Imunofluorescência , Células Endoteliais da Veia Umbilical Humana/imunologia , Células Endoteliais da Veia Umbilical Humana/patologia , Células Endoteliais da Veia Umbilical Humana/virologia , Humanos , Inflamação/imunologia , Inflamação/patologia , Inflamação/virologia , Neovascularização Patológica , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Sarcoma de Kaposi/imunologia , Sarcoma de Kaposi/patologia , Proteínas Virais/genética , Proteínas Virais/imunologia , Proteínas Virais/metabolismo
18.
Toxicol Lett ; 229(1): 229-39, 2014 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-24974766

RESUMO

Trichloroethylene (TCE) is a major occupational health hazard and causes occupational medicamentosa-like dermatitis (OMLDT) and liver damage. Recent evidence suggests immune response as a distinct mode of action for TCE-induced liver damage. This study aimed to explore the role of the key complement activation product C3a and its receptor C3aR in TCE-induced immune liver injury. A mouse model of skin sensitization was induced by TCE in the presence and absence of the C3aR antagonist SB 290157. Liver function was evaluated by alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in conjunction with histopathological characterizations. C3a and C3aR were detected by immunohistochemistry and C5b-9 was assessed by immunofluorescence. IFN-γ and IL4 expressions were determined by flow cytometry and ELISA. The total sensitization rate was 44.1%. TCE sensitization caused liver cell necrosis and inflammatory infiltration, elevated serum ALT and AST, expression of C3a and C3aR, and deposition of C5b-9 in the liver. IFN-γ and IL-4 expressions were up-regulated in spleen mononuclear cells and their serum levels were also increased. Pretreatment with SB 290157 resulted in more inflammatory infiltration in the liver, higher levels of AST, reduced C3aR expression on Kupffer cells, and decreased IL-4 levels while IFN-γ remained unchanged. These data demonstrate that blocking of C3a binding to C3aR reduces IL4, shifts IFN-γ and IL-4 balance, and aggravates TCE-sensitization induced liver damage. These findings reveal a novel mechanism whereby modulation of Th2 response by C3a binding to C3a receptor contributes to immune-mediated liver damage by TCE exposure.


Assuntos
Doença Hepática Induzida por Substâncias e Drogas/patologia , Complemento C3a/metabolismo , Receptores de Complemento/metabolismo , Solventes/toxicidade , Células Th2/efeitos dos fármacos , Tricloroetileno/toxicidade , Animais , Arginina/análogos & derivados , Arginina/farmacologia , Compostos Benzidrílicos/farmacologia , Ativação do Complemento/efeitos dos fármacos , Complemento C5b/biossíntese , Feminino , Citometria de Fluxo , Imunofluorescência , Imuno-Histoquímica , Interferon gama/metabolismo , Interleucina-4/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Testes de Função Hepática , Camundongos , Camundongos Endogâmicos BALB C , Receptores de Complemento/antagonistas & inibidores , Pele/metabolismo , Pele/patologia
19.
Blood ; 123(24): 3733-8, 2014 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-24695849

RESUMO

Atypical hemolytic uremic syndrome (aHUS) is characterized by dysregulated complement activity, the development of a thrombotic microangiopathy (TMA), and widespread end organ injury. aHUS remains a clinical diagnosis without an objective laboratory test to confirm the diagnosis. We performed a retrospective analysis of 103 patients enrolled in the Ohio State University TTP/aHUS Registry presenting with an acute TMA. Nineteen patients were clinically categorized as aHUS based on the following criteria: (1) platelet count <100 × 10(9)/L, (2) serum creatinine >2.25 mg/dL, and (3) a disintegrin and metalloprotease with thrombospondin type 1 motif, 13 (ADAMTS13) activity >10%. Sixteen of 19 patients were treated with plasma exchange (PEX) therapy, with 6/16 (38%) responding to PEX. Nine patients were treated with eculizumab with 7/9 (78%) responding to therapy. In contrast to thrombotic thrombocytopenic purpura (TTP) patients, no aHUS patients demonstrated ultralarge von Willebrand factor multimers at presentation. Median markers of generalized complement activation (C3a), alternative pathway (Bb), classical/lectin pathway (C4d), and terminal complement activation (C5a and C5b-9) were increased in the plasma of these 19 patients. Compared with a cohort of ADAMTS13-deficient TTP patients (n = 38), C5a and C5-9 were significantly higher in the 19 patients clinically characterized as aHUS, suggesting that pretreatment measurements of complement biomarkers C5a and C5b-9 may confirm the diagnosis of aHUS and differentiate it from TTP.


Assuntos
Ativação do Complemento , Síndrome Hemolítico-Urêmica/diagnóstico , Púrpura Trombocitopênica Trombótica/diagnóstico , Adolescente , Adulto , Idoso , Síndrome Hemolítico-Urêmica Atípica , Biomarcadores/análise , Biomarcadores/sangue , Complemento C5a/análise , Complemento C5b/análise , Diagnóstico Diferencial , Feminino , Síndrome Hemolítico-Urêmica/sangue , Humanos , Masculino , Pessoa de Meia-Idade , Púrpura Trombocitopênica Trombótica/sangue , Estudos Retrospectivos , Adulto Jovem
20.
J Immunol ; 192(5): 2339-48, 2014 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-24489093

RESUMO

Traumatic brain injury (TBI) is the leading cause of disability and death in young adults. The secondary neuroinflammation and neuronal damage that follows the primary mechanical injury is an important cause of disability in affected people. The membrane attack complex (MAC) of the complement system is detected in the traumatized brain early after TBI; however, its role in the pathology and neurologic outcome of TBI has not yet been investigated. We generated a C6 antisense oligonucleotide that blocks MAC formation by inhibiting C6, and we compared its therapeutic effect to that of Ornithodoros moubata complement inhibitor (OmCI), a known inhibitor of C5 activation that blocks generation of the anaphylatoxin C5a and C5b, an essential component of MAC. Severe closed head injury in mice induced abundant MAC deposition in the brain. Treatment with C6 antisense reduced C6 synthesis (85%) and serum levels (90%), and inhibited MAC deposition in the injured brain (91-96%). Treatment also reduced accumulation of microglia/macrophages (50-88%), neuronal apoptosis, axonal loss and weight loss (54-93%), and enhanced neurologic performance (84-92%) compared with placebo-treated controls after injury. These data provide the first evidence, to our knowledge, that inhibition of MAC formation in otherwise complement-sufficient animals reduces neuropathology and promotes neurologic recovery after TBI. Given the importance of maintaining a functional complement opsonization system to fight infections, a critical complication in TBI patients, inhibition of the MAC should be considered to reduce posttraumatic neurologic damage. This work identifies a novel therapeutic target for TBI and will guide the development of new therapy for patients.


Assuntos
Proteínas de Artrópodes/farmacologia , Axônios/imunologia , Lesões Encefálicas/tratamento farmacológico , Proteínas de Transporte/farmacologia , Complexo de Ataque à Membrana do Sistema Complemento/antagonistas & inibidores , Macrófagos/imunologia , Oligodesoxirribonucleotídeos Antissenso/farmacologia , Animais , Axônios/patologia , Lesões Encefálicas/imunologia , Lesões Encefálicas/patologia , Complemento C5a/antagonistas & inibidores , Complemento C5a/imunologia , Complemento C5b/antagonistas & inibidores , Complemento C5b/imunologia , Complexo de Ataque à Membrana do Sistema Complemento/imunologia , Feminino , Macrófagos/patologia , Camundongos , Camundongos Endogâmicos BALB C , Microglia/imunologia , Microglia/patologia , Recuperação de Função Fisiológica/efeitos dos fármacos , Recuperação de Função Fisiológica/imunologia
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