RESUMO
To clarify the role of the C5a/C5aR (C5a receptor) and C5b-9 pathways in macrovascular thrombosis (MAT) and renal microthrombosis (MIT), 73 renal biopsy-proven complement-mediated thrombotic microangiopathy (C-TMA) patients were enrolled; 9 patients with pure MAT and 13 patients with pure MIT were selected for further study. Twenty-five external C-TMA patients were selected as the validation cohort. Plasma C5a and sC5b-9 (soluble C5b-9) levels were significantly higher in patients with MAT than in those with MIT (P = 0.008, P = 0.041, respectively). The mean optical density of C5aR1 in the kidney was significantly higher in MAT patients than in those with MIT (P < 0.001). Both urinary sC5b-9 levels (MIT: P < 0.001, MAT: P = 0.004) and renal deposition of C5b-9 (MIT: P < 0.001, MAT: P = 0.001) were significantly higher in C-TMA patients compared to normal control, but were similar between MAT and MIT groups. In the correlation analysis within 22C-TMA patients, urinary sC5b-9 levels and renal deposition of C5b-9 were positively correlated to renal MIT formation (P = 0.009 and P = 0.031, respectively). Furthermore, the renal citrullinated histone H3 (CitH3)- and neutrophil elastase (NE)-positive area ratios were both significantly higher in the MAT group than in the MIT group (P = 0.006 and P = 0.020, respectively). Therefore, the local C5b-9 and C5a/C5aR1 pathways might have differential contributions to MIT and MAT formation in the disease.
Assuntos
Trombose , Microangiopatias Trombóticas , Humanos , Ativação do Complemento , Complexo de Ataque à Membrana do Sistema Complemento/metabolismo , Proteínas do Sistema Complemento , Receptor da Anafilatoxina C5a , Complemento C5bRESUMO
Immunological/inflammatory factors are implicated in the development of psychosis. Complement is a key driver of inflammation; however, it remains unknown which factor is better at predicting the onset of psychosis. This study aimed to compare the alteration and predictive performance of inflammation and complement in individuals at clinical high risk (CHR). We enrolled 49 individuals at CHR and 26 healthy controls (HCs). Twenty-five patients at CHR had converted to psychosis (converter) by the 3-year follow-up. Inflammatory cytokines, including interleukin (IL)-1ß, 6, 8, 10, tumor necrosis factor-alpha (TNF-alpha), macrophage colony-stimulating factor levels, and complement proteins (C1q, C2, C3, C3b, C4, C4b, C5, C5a, factor B, D, I, H) were measured by enzyme-linked immunosorbent assay at baseline. Except for TNF- alpha, none of the inflammatory cytokines reached a significant level in either the comparison of CHR individuals and HC or between CHR-converters and non-converters. The C5, C3, D, I, and H levels were significantly lower (C5, p = 0.006; C3, p = 0.009; D, p = 0.026; I, p = 0.016; H, p = 0.019) in the CHR group than in the HC group. Compared to non-converters, converters had significantly lower levels of C5 (p = 0.012) and C5a (p = 0.007). None of the inflammatory factors, but many complement factors, showed significant correlations with changes in general function and symptoms. None of the inflammatory markers, except for C5a and C5, were significant in the discrimination of conversion outcomes in CHR individuals. Our results suggest that altered complement levels in the CHR population are more associated with conversion to psychosis than inflammatory factors. Therefore, an activated complement system may precede the first-episode of psychosis and contribute to neurological pathogenesis at the CHR stage.
Assuntos
Proteínas do Sistema Complemento , Transtornos Psicóticos , Humanos , Citocinas/sangue , Citocinas/química , Inflamação/metabolismo , Transtornos Psicóticos/sangue , Transtornos Psicóticos/diagnóstico , Fatores de Risco , Fator de Necrose Tumoral alfa , Proteínas do Sistema Complemento/química , Complemento C1q/química , Complemento C3b/química , Complemento C4b/química , Complemento C5b/químicaRESUMO
The Membrane Attack Complex (MAC) is responsible for forming large ß-barrel channels in the membranes of pathogens, such as gram-negative bacteria. Off-target MAC assembly on endogenous tissue is associated with inflammatory diseases and cancer. Accordingly, a human C5b-9 specific antibody, aE11, has been developed that detects a neoepitope exposed in C9 when it is incorporated into the C5b-9 complex, but not present in the plasma native C9. For nearly four decades aE11 has been routinely used to study complement, MAC-related inflammation, and pathophysiology. However, the identity of C9 neoepitope remains unknown. Here, we determined the cryo-EM structure of aE11 in complex with polyC9 at 3.2 Å resolution. The aE11 binding site is formed by two separate surfaces of the oligomeric C9 periphery and is therefore a discontinuous quaternary epitope. These surfaces are contributed by portions of the adjacent TSP1, LDLRA, and MACPF domains of two neighbouring C9 protomers. By substituting key antibody interacting residues to the murine orthologue, we validated the unusual binding modality of aE11. Furthermore, aE11 can recognise a partial epitope in purified monomeric C9 in vitro, albeit weakly. Taken together, our results reveal the structural basis for MAC recognition by aE11.
Assuntos
Complemento C9 , Complexo de Ataque à Membrana do Sistema Complemento , Humanos , Animais , Camundongos , Complexo de Ataque à Membrana do Sistema Complemento/metabolismo , Complemento C5b , Complemento C9/química , Complemento C9/metabolismo , Proteínas do Sistema Complemento/metabolismo , EpitoposRESUMO
Patients with COVID-19 who require ICU admission might have the cytokine storm. It is a state of out-of-control release of a variety of inflammatory cytokines. The molecular mechanism of the cytokine storm has not been explored extensively yet. The attachment of SARS-CoV-2 spike glycoprotein with angiotensin-converting enzyme 2 (ACE2), as its cellular receptor, triggers complex molecular events that leads to hyperinflammation. Four molecular axes that may be involved in SARS-CoV-2 driven inflammatory cytokine overproduction are addressed in this work. The virus-mediated down-regulation of ACE2 causes a burst of inflammatory cytokine release through dysregulation of the renin-angiotensin-aldosterone system (ACE/angiotensin II/AT1R axis), attenuation of Mas receptor (ACE2/MasR axis), increased activation of [des-Arg9]-bradykinin (ACE2/bradykinin B1R/DABK axis), and activation of the complement system including C5a and C5b-9 components. The molecular clarification of these axes will elucidate an array of therapeutic strategies to confront the cytokine storm in order to prevent and treat COVID-19 associated acute respiratory distress syndrome.
Assuntos
Betacoronavirus/metabolismo , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/metabolismo , Citocinas/metabolismo , Inflamação/metabolismo , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/imunologia , Pneumonia Viral/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Enzima de Conversão de Angiotensina 2 , Betacoronavirus/patogenicidade , Bradicinina/metabolismo , COVID-19 , Complemento C5a/imunologia , Complemento C5a/metabolismo , Complemento C5b/imunologia , Complemento C5b/metabolismo , Infecções por Coronavirus/enzimologia , Humanos , Inflamação/enzimologia , Inflamação/imunologia , Modelos Moleculares , Pandemias , Pneumonia Viral/enzimologia , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Sistema Renina-Angiotensina/imunologia , SARS-CoV-2RESUMO
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/metabolismoRESUMO
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
Injúria Renal Aguda/imunologia , Rim/fisiologia , NF-kappa B/metabolismo , Injúria 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/toxicidadeRESUMO
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/metabolismoRESUMO
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/farmacologiaRESUMO
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/patologiaRESUMO
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/imunologiaRESUMO
Objetivos: 1) Identificar las variables que se asocian con los niveles urinarios de MBL, C4d y C5b-9 en enfermos con nefropatía IgA idiopática. 2) Analizar si los niveles urinarios de MBL o C4d son útiles para identificar la presencia de depósitos mesangiales de C4d/MBL. Pacientes y método: Se estudió a 96 enfermos con nefropatía IgA primaria. Se registraron las variables demográficas, clínicas y bioquímicas en el momento del diagnóstico. Las lesiones renales se cuantificaron mediante la clasificación de Oxford. En las biopsias, se realizaron tinciones inmunohistoquímicas para MBL, properdina, C4d, y C5b-9. En orina, se determinó el nivel de properdina, MBL, C4d y C5b-9. Resultados: Los predictores independientes de los niveles de C4d y MBL en orina fueron el depósito mesangial de cada una de ellas y, en menor grado, la proteinuria. Los predictores independientes de los niveles urinarios de C5b-9 fueron los niveles de MBL y properdina, y la proteinuria. La excreción urinaria de C4d tuvo una sensibilidad del 90% (IC 95%: 58,7-99) y una especificidad del 73% (IC 95%: 54-87) para la detección de depósitos mesangiales de C4d y el nivel de MBL tuvo una sensibilidad del 83,9% (IC 95%: 62-95) y una especificidad del 81,6% (IC 95%: 65-92) para identificar depósitos mesangiales de MBL. Conclusión: El principal predictor de la concentración urinaria de C4d y MBL es la presencia de depósitos mesangiales de ellas. La MBL podría contribuir a la activación del complemento en la luz tubular a través de la vía de las lectinas. Los niveles urinarios de MBL y C4d podrían ser biomarcadores sensibles y específicos para la identificación de los enfermos que presentan depósitos mesangiales de MBL o C4d (AU)
Objectives: 1. To identify the variables that are associated with urinary levels of properdin, MBL, C4d, and C5b-9 in patients with idiopathic IgA nephropathy. 2. To analyse whether urinary levels of MBL and/or C4d are useful for identifying the presence of mesangial deposits of C4d/MBL. Patients and method: A total of 96 patients with IgA nephropathy were studied. Demographic, clinical and biochemical variables were recorded at the time of diagnosis. Renal lesions were quantified using the Oxford classification. Immunohistochemical staining for MBL, MASP-2, properdin, C4d, and C5b-9 was performed in kidney biopsies, and in urine, the levels of properdin, MBL, C4d and C5b-9 were determined. Results: In multivariate analysis, the independent predictors of C4d and MBL levels in urine were the mesangial deposits of each protein and, to a lesser extent, the urinary protein excretion. The independent predictors of urinary levels of C5b-9 were MBL properdin and proteinuria. Urinary excretion of C4d had a sensitivity of 90% (95% CI: 58,7 to 99) and a specificity of 73% (95% CI: 54-87) for detecting mesangial C4d deposits, and the level of MBL had a sensitivity of 83.9% (95% CI: 62-95) and a specificity of 81.6% (95% CI: 65-92) for identifying mesangial deposits of MBL. Conclusion: The main predictor of urinary concentration of C4d and MBL was the presence of their respective mesangial deposits. Urine MBL may contribute to complement activation in the tubular luz through the lectin pathway. Urinary levels of MBL and C4d could be sensitive and specific biomarkers for the identification of patients with mesangial deposits of MBL and C4d (AU)
Assuntos
Humanos , Glomerulonefrite por IGA/fisiopatologia , Ativação do Complemento/fisiologia , Proteína de Ligação ao Complemento C4b/análise , Complemento C5b/análise , Biópsia/métodos , Properdina/análise , Imuno-Histoquímica/métodos , Proteinúria/epidemiologia , Lectina de Ligação a Manose/análiseRESUMO
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/patologiaRESUMO
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 , Imageamento 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/metabolismoRESUMO
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/fisiopatologiaRESUMO
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 immunoï¬uorescence. mRNA and protein levels of B1R and B2R were assessed by real-time qPCR and Western blot. As expected, numerous inflammatory cell inï¬ltration 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
Injúria 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/metabolismoRESUMO
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 ProteínaRESUMO
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 , MasculinoRESUMO
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 EspecificidadeRESUMO
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 , NecroseRESUMO
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.