Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 638
Filtrar
1.
Toxicol Lett ; 317: 110-119, 2019 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-31618666

RESUMO

Trichloroethylene (TCE), a commonly used industrial solvent and degreasing agent, is known to cause trichloroethylene hypersensitivity syndrome (THS) with multi-system damage, including skin, liver and kidney. Clinical evidence have shown that the kidney injury occurs in THS and our previous studies suggested that the terminal complement complex C5b-9 deposited in impaired renal tubules induced by TCE with unclear mechanisms. In the present study, we questioned whether activation of the complement system with renal deposition of C5b-9 contributes to TCE-induced kidney injury in THS. We established a BALB/c mouse model of TCE sensitization with or without pretreatment of exogenous CD59, a C5b-9 inhibitory protein. H&E staining, PAS staining, and biochemical detection of urinary proteins were performed to assess renal function. Deposition of C5b-9 and expression of CD59 were evaluated by immunohistochemistry. Sub-lytic effects of C5b-9 in tubular epithelial cells were assessed by lactate dehydrogenase (LDH) cytotoxicity assay. Expression of endocytosis receptors megalin and cubilin on proximal tubules were assessed by immunofluorescence and qRT-PCR. We found that TCE sensitization induced structural and functional changes of renal tubules in mice, associated with the deposition of sub-lytic C5b-9 on proximal tubular epithelial cells. TCE sensitization decreased proximal tubule uptake of filtered proteins and renal expression of megalin and cubilin, phenotypes that were attenuated by pretreatment with exogenous CD59. Overall, our findings reveal a novel mechanism underlying sub-lytic C5b-9 acting on megalin and cubilin, contributes to the renal tubules damage by TCE exposure.


Assuntos
Complexo de Ataque à Membrana do Sistema Complemento/metabolismo , Endocitose , Hipersensibilidade/metabolismo , Nefropatias/metabolismo , Túbulos Renais Proximais/metabolismo , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Receptores de Superfície Celular/metabolismo , Tricloroetileno , Animais , Células Cultivadas , Ativação do Complemento , Complexo de Ataque à Membrana do Sistema Complemento/imunologia , Modelos Animais de Doenças , Feminino , Hipersensibilidade/imunologia , Hipersensibilidade/patologia , Nefropatias/induzido quimicamente , Nefropatias/imunologia , Nefropatias/patologia , Túbulos Renais Proximais/imunologia , Túbulos Renais Proximais/patologia , Camundongos Endogâmicos BALB C , Transporte Proteico
2.
Thromb Haemost ; 119(9): 1433-1440, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31266080

RESUMO

Transplant-associated thrombotic microangiopathy (TA-TMA) is a severe and life-threatening complication of hematopoietic cell transplantation (HCT) that often coincides with graft-versus-host-disease (GVHD). Although endothelial damage seems to be the common denominator for both disorders, the role of complement system, neutrophils, and coagulation has not been clarified. In an effort to distinguish the pathogenesis of TA-TMA from GVHD, we evaluated markers of complement activation, neutrophil extracellular trap (NET) release, endothelial damage, and activation of coagulation cascade in the circulation of patients with these two disorders, as well as control HCT recipients without TA-TMA or GVHD. We observed that the terminal complement product C5b-9 levels, the levels of markers of NET formation, and thrombin-antithrombin complex levels were significantly increased in the TA-TMA group compared with patients without complications, whereas there was no significant difference between the GVHD and the control group. On the other hand, the levels of circulating thrombomodulin, an endothelial damage marker, were significantly increased in both TA-TMA and GVHD patients. These findings propose a role for the interplay between complement system, neutrophil activation through NET release, and activation of the coagulation cascade in TA-TMA.


Assuntos
Endotélio Vascular/patologia , Armadilhas Extracelulares/metabolismo , Doença Enxerto-Hospedeiro/imunologia , Transplante de Células-Tronco Hematopoéticas , Neutrófilos/imunologia , Complicações Pós-Operatórias/imunologia , Microangiopatias Trombóticas/imunologia , Adulto , Idoso , Antitrombina III , Biomarcadores/metabolismo , Coagulação Sanguínea , Estudos de Casos e Controles , Ativação do Complemento , Complexo de Ataque à Membrana do Sistema Complemento/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Peptídeo Hidrolases/sangue , Trombomodulina/sangue , Microangiopatias Trombóticas/etiologia , Adulto Jovem
3.
Nat Commun ; 10(1): 3325, 2019 07 25.
Artigo em Inglês | MEDLINE | ID: mdl-31346171

RESUMO

Serum resistance is a poorly understood but common trait of some difficult-to-treat pathogenic strains of bacteria. Here, we report that glycine, serine and threonine catabolic pathway is down-regulated in serum-resistant Escherichia coli, whereas exogenous glycine reverts the serum resistance and effectively potentiates serum to eliminate clinically-relevant bacterial pathogens in vitro and in vivo. We find that exogenous glycine increases the formation of membrane attack complex on bacterial membrane through two previously unrecognized regulations: 1) glycine negatively and positively regulates metabolic flux to purine biosynthesis and Krebs cycle, respectively. 2) α-Ketoglutarate inhibits adenosine triphosphate synthase, which in together promote the formation of cAMP/CRP regulon to increase the expression of complement-binding proteins HtrE, NfrA, and YhcD. The results could lead to effective strategies for managing the infection with serum-resistant bacteria, an especially valuable approach for treating individuals with weak acquired immunity but a normal complement system.


Assuntos
Proteínas do Sistema Complemento/imunologia , Infecções por Escherichia coli/imunologia , Escherichia coli/metabolismo , Glicina/metabolismo , Serina/metabolismo , Soro/química , Treonina/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Chaperoninas/genética , Chaperoninas/metabolismo , Ciclo do Ácido Cítrico , Complexo de Ataque à Membrana do Sistema Complemento/genética , Complexo de Ataque à Membrana do Sistema Complemento/metabolismo , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Humanos , Purinas/biossíntese
4.
Biochem Soc Trans ; 47(3): 801-810, 2019 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-31209154

RESUMO

Members of the membrane attack complex/perforin-like (MACPF) protein superfamily have long captured interest because of their unique ability to assemble into large oligomeric pores on the surfaces of cells. The best characterised of these act in vertebrate immunity where they function to deliver pro-apoptotic factors or induce the cytolysis and death of targeted cells. Less appreciated, however, is that rather than causing cell death, MACPF proteins have also evolved to control cellular signalling pathways and influence developmental programmes such as pattern formation and neurogenesis. Torso-like (Tsl) from the fruit fly Drosophila, for example, functions to localise the activity of a growth factor for patterning its embryonic termini. It remains unclear whether these developmental proteins employ an attenuated form of the classical MACPF lytic pore, or if they have evolved to function via alternative mechanisms of action. In this minireview, we examine the evidence that links pore-forming MACPF proteins to the control of growth factor and cytokine signalling. We will then attempt to reconcile how the MACPF domain may have been repurposed during evolution for developmental events rather than cell killing.


Assuntos
Complexo de Ataque à Membrana do Sistema Complemento/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Transdução de Sinais , Animais , Drosophila/metabolismo , Humanos
5.
Nat Commun ; 10(1): 2247, 2019 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-31113953

RESUMO

Complement promotes vascular inflammation in transplant organ rejection and connective tissue diseases. Here we identify ZFYVE21 as a complement-induced Rab5 effector that induces non-canonical NF-κB in endothelial cells (EC). In response to membrane attack complexes (MAC), ZFYVE21 is post-translationally stabilized on MAC+Rab5+ endosomes in a Rab5- and PI(3)P-dependent manner. ZFYVE21 promotes SMURF2-mediated polyubiquitinylation and proteasome-dependent degradation of endosome-associated PTEN to induce vesicular enrichment of PI(3,4,5)P3 and sequential recruitment of activated Akt and NF-κB-inducing kinase (NIK). Pharmacologic alteration of cellular phosphoinositide content with miltefosine reduces ZFYVE21 induction, EC activation, and allograft vasculopathy in a humanized mouse model. ZFYVE21 induction distinctly occurs in response to MAC and is detected in human renal and synovial tissues. Our data identifies ZFYVE21 as a Rab5 effector, defines a Rab5-ZFYVE21-SMURF2-pAkt axis by which it mediates EC activation, and demonstrates a role for this pathway in complement-mediated conditions.


Assuntos
Proteínas de Transporte/metabolismo , Endossomos/metabolismo , Rejeição de Enxerto/patologia , NF-kappa B/metabolismo , Vasculite/patologia , Aloenxertos/patologia , Animais , Linhagem Celular , Complexo de Ataque à Membrana do Sistema Complemento/metabolismo , Vasos Coronários/patologia , Vasos Coronários/transplante , Modelos Animais de Doenças , Feminino , Células Endoteliais da Veia Umbilical Humana , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas de Membrana , Camundongos , Camundongos SCID , Fosfatos de Fosfatidilinositol/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas rab5 de Ligação ao GTP/metabolismo
6.
Nat Commun ; 10(1): 2066, 2019 05 06.
Artigo em Inglês | MEDLINE | ID: mdl-31061395

RESUMO

The membrane attack complex (MAC) is a hetero-oligomeric protein assembly that kills pathogens by perforating their cell envelopes. The MAC is formed by sequential assembly of soluble complement proteins C5b, C6, C7, C8 and C9, but little is known about the rate-limiting steps in this process. Here, we use rapid atomic force microscopy (AFM) imaging to show that MAC proteins oligomerize within the membrane, unlike structurally homologous bacterial pore-forming toxins. C5b-7 interacts with the lipid bilayer prior to recruiting C8. We discover that incorporation of the first C9 is the kinetic bottleneck of MAC formation, after which rapid C9 oligomerization completes the pore. This defines the kinetic basis for MAC assembly and provides insight into how human cells are protected from bystander damage by the cell surface receptor CD59, which is offered a maximum temporal window to halt the assembly at the point of C9 insertion.


Assuntos
Antígenos CD59/metabolismo , Membrana Celular/ultraestrutura , Complemento C9/metabolismo , Complexo de Ataque à Membrana do Sistema Complemento/metabolismo , Multimerização Proteica , Membrana Celular/metabolismo , Complemento C5/metabolismo , Complemento C8/metabolismo , Humanos , Cinética , Microscopia de Força Atômica/métodos , Imagem Individual de Molécula/métodos
7.
Toxins (Basel) ; 11(5)2019 04 26.
Artigo em Inglês | MEDLINE | ID: mdl-31027358

RESUMO

Bee venom (BV)-a complex mixture of peptides and toxic proteins including phospholipase A2 and melittin-promotes blood clotting. In this study, we investigated the anti-atopic properties of BV and the mechanism associated with its regulation of the complement system. BV treatment upregulated the mRNA and protein levels of CD55 in THP-1 cells. Further experiments revealed that the phosphorylation of ERK was associated with upregulation of CD55. A complement-dependent cytotoxicity assay and a bacteria-killing assay showed that BV inactivated the complement system through the induction of CD55. The serum levels of C3 convertase (C3C) and Membrane attack complex (MAC) increased, while CD55 decreased in mice with AD-like lesions from DNCB treatment. However, the levels were inverted when the AD-like mice were treated with BV using subcutaneous injection, and we observed that the AD symptoms were alleviated. BV is often used to treat AD but its mechanism has not been elucidated. Here, we suggest that BV alleviates AD through the inactivation of the complement system, especially by the induction of CD55.


Assuntos
Anti-Inflamatórios/uso terapêutico , Venenos de Abelha/uso terapêutico , Antígenos CD55/metabolismo , Dermatite Atópica/tratamento farmacológico , Animais , Anti-Inflamatórios/farmacologia , Venenos de Abelha/farmacologia , Antígenos CD55/genética , Linhagem Celular , Convertases de Complemento C3-C5/metabolismo , Complexo de Ataque à Membrana do Sistema Complemento/metabolismo , Dermatite Atópica/genética , Dermatite Atópica/metabolismo , Escherichia coli/efeitos dos fármacos , Humanos , Masculino , Camundongos Endogâmicos BALB C , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Regulação para Cima/efeitos dos fármacos
8.
Mol Cell Endocrinol ; 490: 57-67, 2019 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-30981734

RESUMO

In the human placenta, extravillous trophoblasts (EVTs) invade maternal decidual tissues (interstitial trophoblasts) and maternal spiral arteries (endovascular trophoblasts). Although endovascular trophoblasts are directly exposed to maternal blood containing complement components, they are not eliminated by complement-dependent cytotoxicity (CDC). In this study, we investigated the expression and possible function of CD59, one of the membrane-bound complement regulators, in EVTs. Immunohistochemistry of early embryo implantation sites revealed that CD59 was hardly expressed on interstitial trophoblasts, whereas it was intensely expressed on endovascular trophoblasts. Using the human EVT-like cell line Swan71, we established CD59-silencing Swan71 cells (Sw_CD59sh) and non-silencing control Swan71 cells (Sw_CTRsh). In vitro cell apoptosis assay showed that Sw_CD59sh cells were significantly more susceptible to CDC as compared to Sw_CTRsh. Our results suggest that CD59 confers some protection against maternal complement attack to the endovascular trophoblasts.


Assuntos
Antígenos CD59/metabolismo , Proteínas do Sistema Complemento/metabolismo , Citotoxicidade Imunológica , Trofoblastos/metabolismo , Hipóxia Celular , Linhagem Celular , Complexo de Ataque à Membrana do Sistema Complemento/metabolismo , Decídua/metabolismo , Implantação do Embrião/genética , Feminino , Regulação da Expressão Gênica , Humanos , Gravidez
9.
Pediatr Dev Pathol ; 22(4): 356-364, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30722724

RESUMO

Autopsy reports of 78 stillbirths and early infant deaths (up to age 8 weeks) were reviewed to investigate the prevalence of extrahepatic nonreticuloendothelial siderosis (EHNRS) in the context of neonatal liver failure. Of these, 10 liveborns (12.8%), M:F 3:2, with mean gestational age 37.6 weeks (range: 35-39) and mean age at the time of demise 19.1 days (range: 7-42), showed significant liver injury: infection (n = 7, viral > fungal), congenital malformations (n = 2), and ischemia (n = 1). None had maternal history of gestational alloimmune liver disease (GALD) or previous fetal/neonatal death due to liver failure. Seven of 10 cases (70%) showed EHNRS: pancreas (n = 6), kidneys (n = 4), thyroid and adrenal glands (n = 3), and bronchial glands and heart (n = 2). Iron deposition was most frequent in the pancreas (60%), most diffuse in the kidneys, and seen in at least 2 organs, with pancreas and kidney being the most frequent combination. Hepatic C5b-9 expression was variable (1+ to 4+) except 1 case (100% necrosis). The duration of illness and the mean age at the time of demise tended to be higher in those with EHNRS. In summary, hepatic and EHNRS, with or without C5b-9 expression, are not specific for GALD. Other causes of liver failure should be investigated as clinically and pathologically appropriate.


Assuntos
Complexo de Ataque à Membrana do Sistema Complemento/metabolismo , Morte Fetal/etiologia , Doenças do Recém-Nascido/etiologia , Ferro/metabolismo , Falência Hepática/etiologia , Siderose/etiologia , Feminino , Humanos , Imuno-Histoquímica , Recém-Nascido , Doenças do Recém-Nascido/patologia , Fígado/metabolismo , Fígado/patologia , Falência Hepática/complicações , Falência Hepática/patologia , Assistência Perinatal , Gravidez , Estudos Retrospectivos , Siderose/patologia , Natimorto
10.
Invest Ophthalmol Vis Sci ; 60(2): 461-472, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30707219

RESUMO

Purpose: Multiple evidence lines support Bruch's membrane lipid deposition as a major precursor of soft drusen and age-related macular degeneration as including a potentially treatable atherosclerosis-like progression in the subretinal pigment epithelium (RPE)-basal lamina space. We evaluated the effect of anti-inflammatory, antiatherogenic peptide L-4F on Bruch's membrane of aged nonhuman primates in a dose-escalating study. Methods: Macaca fascicularis ≥20 years of age evaluated by color fundus photography and optical coherence tomography received monocular intravitreal injections of L-4F (n = 7) or a placebo-scrambled peptide (n = 2) in 6 doses of 25 to 175 µg over 6 months. Eyes were processed for detection and masked semiquantitative assessment of macular Bruch's membrane neutral lipid (oil red O staining), esterified cholesterol (filipin histochemistry), membrane attack complex (immunofluorescence), and paramacular thickness (transmission electron microscopy). Results: Bruch's membrane neutral lipid, esterified cholesterol, and membrane attack complex were cleared and ultrastructure was improved in L-4F-injected eyes, compared to placebo-injected eyes. Fellow eyes were also affected to the same degree as the injected eyes. Punctate yellow fundus lesions without corresponding RPE elevations on optical coherence tomography correlated to RPE lipoidal degeneration (engorgement with lipid droplets), which was unchanged by this treatment. Conclusions: Clinical-stage apolipoprotein A-I mimetic peptide L-4F, delivered intravitreally in repeated doses, produced a substantial pharmacologic reduction of Bruch's membrane lipid and restoration of ultrastructure in a nonhuman primate model that exhibits an important precursor of soft drusen, if not soft drusen themselves.


Assuntos
Envelhecimento/fisiologia , Lâmina Basilar da Corioide/efeitos dos fármacos , Lipídeos de Membrana/metabolismo , Peptídeos/farmacologia , Animais , Anti-Inflamatórios não Esteroides/administração & dosagem , Anti-Inflamatórios não Esteroides/farmacologia , Compostos Azo/metabolismo , Lâmina Basilar da Corioide/metabolismo , Lâmina Basilar da Corioide/ultraestrutura , Complexo de Ataque à Membrana do Sistema Complemento/metabolismo , Relação Dose-Resposta a Droga , Filipina/metabolismo , Fundo de Olho , Histocitoquímica/métodos , Injeções Intravítreas , Macaca fascicularis , Microscopia Eletrônica de Transmissão , Imagem Multimodal , Peptídeos/administração & dosagem , Fotografação , Tomografia de Coerência Óptica
11.
EMBO J ; 38(4)2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30643019

RESUMO

The immune system kills bacteria by the formation of lytic membrane attack complexes (MACs), triggered when complement enzymes cleave C5. At present, it is not understood how the MAC perturbs the composite cell envelope of Gram-negative bacteria. Here, we show that the role of C5 convertase enzymes in MAC assembly extends beyond the cleavage of C5 into the MAC precursor C5b. Although purified MAC complexes generated from preassembled C5b6 perforate artificial lipid membranes and mammalian cells, these components lack bactericidal activity. In order to permeabilize both the bacterial outer and inner membrane and thus kill a bacterium, MACs need to be assembled locally by the C5 convertase enzymes. Our data indicate that C5b6 rapidly loses the capacity to form bactericidal pores; therefore, bacterial killing requires both in situ conversion of C5 and immediate insertion of C5b67 into the membrane. Using flow cytometry and atomic force microscopy, we show that local assembly of C5b6 at the bacterial surface is required for the efficient insertion of MAC pores into bacterial membranes. These studies provide basic molecular insights into MAC assembly and bacterial killing by the immune system.


Assuntos
Atividade Bactericida do Sangue , Membrana Celular/metabolismo , Convertases de Complemento C3-C5/metabolismo , Complexo de Ataque à Membrana do Sistema Complemento/metabolismo , Bactérias Gram-Negativas/crescimento & desenvolvimento , Hemólise , Permeabilidade da Membrana Celular , Ativação do Complemento , Bactérias Gram-Negativas/metabolismo , Humanos
12.
Ecotoxicol Environ Saf ; 172: 105-113, 2019 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-30685621

RESUMO

Trichloroethylene (TCE) is a major occupational and environmental chemical compound which causes occupational dermatitis medicamentosa-like of TCE with severe liver damage. Our previous studies showed that complement activation was a newly recognized mechanism for TCE-induced liver damage. The objective of this study was to explore the role of the key complement regulatory protein, CD59a, in TCE-induced immune liver injury. We firstly evaluated the changes of CD59a expression in liver tissue and then investigated if the changes were associated with membrane attack complex (MAC) formation, nuclear factor kappa B (NF-κB) activation and liver damage in BALB/c mice model of TCE-induced skin sensitization in the absence or presence of soluble recombinant rat CD59-Cys. The results showed that low expression of CD59a accompanied by MAC deposition in the liver of TCE-sensitized BALB/c mice, which was consistent in time. In addition, activation of NF-κB pathway, upregulation of inflammatory cytokine and liver damage also occured. Additional experiment showed that recombinant rat sCD59-Cys alleviated inflammation and liver damage in TCE-sensitized BALB/c mice. Moreover, recombinant rat sCD59-Cys reduced MAC formation and inhibited NF-κB activation measured by P-IκBα and nuclear NF-κB p65 in the liver of TCE-sensitized BALB/c mice. In conclusion, recombinant rat sCD59-Cys plays a protective role in immune liver injury of TCE-sensitized BALB/c mice.


Assuntos
Antígenos CD59/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/prevenção & controle , Regulação da Expressão Gênica , Tricloroetileno/toxicidade , Animais , Antígenos CD59/genética , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Complexo de Ataque à Membrana do Sistema Complemento/genética , Complexo de Ataque à Membrana do Sistema Complemento/metabolismo , Feminino , Imuno-Histoquímica , Inflamação/induzido quimicamente , Inflamação/prevenção & controle , Camundongos , Camundongos Endogâmicos BALB C , NF-kappa B/genética , NF-kappa B/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Pele/efeitos dos fármacos , Pele/metabolismo
13.
PLoS One ; 14(1): e0209024, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30601845

RESUMO

Chronic lymphocytic leukemia (CLL) is the most common adult leukemia in the Western world. The therapeutic approach to CLL includes chemotherapeutic regimens and immunotherapy. Complement-mediated cytotoxicity, which is one of the mechanisms activated by the therapeutic monoclonal antibodies, depends on the availability and activity of the complement (C) system. The aim was to study the structure of circulating C components and evaluate the importance of C5 structural integrity for C activity in CLL patients. Blood samples were collected from 40 naïve CLL patients and 15 normal controls (NC). The Western blot analysis showed abnormal C5 pattern in some CLL patients, while patterns of C3 and C4 were similar in all subjects. Levels of the C activation markers sC5b-9 and C5a were quantified before and after activation via the classical (CP) and alternative (AP) pathways. In patients with abnormal C5, basal levels of sC5b-9 and C5a were increased while activities of the CP and of the CP C5-convertase, the immediate C5-upstream complex, were decreased compared to NC and to patients with normal C5. The data indicate a link between CP activation and apparent C5 alterations in CLL. This provides a potential prognostic tool that may personalize therapy by identifying a sub-group of CLL patients who display an abnormal C5 pattern, high basal levels of sC5b-9 and C5a, and impaired CP activity, and are likely to be less responsive to immunotherapy due to compromised CP activity.


Assuntos
Convertases de Complemento C3-C5/metabolismo , Leucemia Linfocítica Crônica de Células B/metabolismo , Leucemia Linfoide/metabolismo , Western Blotting , Ativação do Complemento/genética , Ativação do Complemento/fisiologia , Complemento C5a/genética , Complemento C5a/fisiologia , Complexo de Ataque à Membrana do Sistema Complemento/genética , Complexo de Ataque à Membrana do Sistema Complemento/metabolismo , Feminino , Humanos , Leucemia Linfocítica Crônica de Células B/sangue , Leucemia Linfoide/sangue , Masculino , Pessoa de Meia-Idade
14.
Am J Pathol ; 189(1): 147-161, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30339839

RESUMO

The terminal complement complex (TCC) is formed on activation of the complement system, a crucial arm of innate immunity. TCC formation on cell membranes results in a transmembrane pore leading to cell lysis. In addition, sublytic TCC concentrations can modulate various cellular functions. TCC-induced effects may play a role in the pathomechanisms of inflammatory disorders of the bone, including rheumatoid arthritis and osteoarthritis. In this study, we investigated the effect of the TCC on bone turnover and repair. Mice deficient for complement component 6 (C6), an essential component for TCC assembly, and mice with a knockout of CD59, which is a negative regulator of TCC formation, were used in this study. The bone phenotype was analyzed in vivo, and bone cell behavior was analyzed ex vivo. In addition, the mice were subjected to a femur osteotomy. Under homeostatic conditions, C6-deficient mice displayed a reduced bone mass, mainly because of increased osteoclast activity. After femur fracture, the inflammatory response was altered and bone formation was disturbed, which negatively affected the healing outcome. By contrast, CD59-knockout mice only displayed minor skeletal alterations and uneventful bone healing, although the early inflammatory reaction to femur fracture was marginally enhanced. These results demonstrate that TCC-mediated effects regulate bone turnover and promote an adequate response to fracture, contributing to an uneventful healing outcome.


Assuntos
Regeneração Óssea , Complexo de Ataque à Membrana do Sistema Complemento , Fraturas do Fêmur , Consolidação da Fratura , Osteoclastos , Animais , Regeneração Óssea/genética , Regeneração Óssea/imunologia , Antígenos CD59/deficiência , Técnicas de Cultura de Células , Complemento C6/deficiência , Complexo de Ataque à Membrana do Sistema Complemento/genética , Complexo de Ataque à Membrana do Sistema Complemento/imunologia , Complexo de Ataque à Membrana do Sistema Complemento/metabolismo , Eritrócitos/imunologia , Eritrócitos/metabolismo , Eritrócitos/patologia , Fraturas do Fêmur/genética , Fraturas do Fêmur/imunologia , Fraturas do Fêmur/metabolismo , Fraturas do Fêmur/patologia , Consolidação da Fratura/genética , Consolidação da Fratura/imunologia , Inflamação/genética , Inflamação/imunologia , Inflamação/metabolismo , Inflamação/patologia , Masculino , Camundongos , Camundongos Knockout , Osteoclastos/imunologia , Osteoclastos/metabolismo , Osteoclastos/patologia , Ovinos
15.
Transl Stroke Res ; 10(3): 279-286, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30173313

RESUMO

The terminal complement complex C5b-9 plays an important role in acute ischemic stroke (AIS) and carotid atherosclerosis. However, the associations between serum C5b-9, the severity and outcome of AIS, and the stability of carotid plaques have not been well investigated. In this clinical study, 70 patients with AIS and 70 healthy controls were enrolled. Serum C5b-9 levels at 72 h after stroke onset were measured by enzyme-linked immunosorbent assay (ELISA). Infarct size, the National Institutes of Health Stroke Scale (NIHSS), the 90-day modified Rankin Scale (mRS), and carotid plaque and stenosis were evaluated. Serum C5b-9 levels were significantly higher in AIS patients than in healthy controls (p < 0.001) and were correlated with infarction sizes (p = 0.045) and the NIHSS (P = 0.035). Furthermore, 90-day mRS analysis demonstrated that the patients with poor outcomes had higher serum C5b-9 levels than those with good outcomes (P < 0.001). Moreover, serum C5b-9 levels in AIS patients with unstable carotid plaques were much higher than in those with stable carotid plaques (P = 0.009). Multivariate logistic regression indicated that C5b-9 could be an independent risk factor for AIS (P < 0.001) and unstable carotid plaques (P = 0.015). Therefore, complement complex C5b-9 may be a potential biomarker in predicting the severity and outcome, as well as the stability of carotid plaques, in AIS patients.


Assuntos
Isquemia Encefálica/sangue , Estenose das Carótidas/sangue , Complexo de Ataque à Membrana do Sistema Complemento/metabolismo , Acidente Vascular Cerebral/sangue , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/sangue , Isquemia Encefálica/diagnóstico , Estenose das Carótidas/diagnóstico , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fatores de Risco , Acidente Vascular Cerebral/diagnóstico , Resultado do Tratamento
16.
Nat Commun ; 9(1): 5316, 2018 12 14.
Artigo em Inglês | MEDLINE | ID: mdl-30552328

RESUMO

The membrane attack complex (MAC) is one of the immune system's first responders. Complement proteins assemble on target membranes to form pores that lyse pathogens and impact tissue homeostasis of self-cells. How MAC disrupts the membrane barrier remains unclear. Here we use electron cryo-microscopy and flicker spectroscopy to show that MAC interacts with lipid bilayers in two distinct ways. Whereas C6 and C7 associate with the outer leaflet and reduce the energy for membrane bending, C8 and C9 traverse the bilayer increasing membrane rigidity. CryoEM reconstructions reveal plasticity of the MAC pore and demonstrate how C5b6 acts as a platform, directing assembly of a giant ß-barrel whose structure is supported by a glycan scaffold. Our work provides a structural basis for understanding how ß-pore forming proteins breach the membrane and reveals a mechanism for how MAC kills pathogens and regulates cell functions.


Assuntos
Complexo de Ataque à Membrana do Sistema Complemento/química , Complexo de Ataque à Membrana do Sistema Complemento/ultraestrutura , Microscopia Crioeletrônica/métodos , Bicamadas Lipídicas/química , Complemento C6/química , Complemento C6/metabolismo , Complemento C6/ultraestrutura , Complemento C7/química , Complemento C7/metabolismo , Complemento C7/ultraestrutura , Complemento C8/química , Complemento C8/metabolismo , Complemento C8/ultraestrutura , Complemento C9/química , Complemento C9/metabolismo , Complemento C9/ultraestrutura , Complexo de Ataque à Membrana do Sistema Complemento/metabolismo , Humanos , Processamento de Imagem Assistida por Computador , Bicamadas Lipídicas/metabolismo , Lipossomos , Modelos Moleculares , Polissacarídeos/metabolismo , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Análise Espectral/métodos
17.
Obstet Gynecol ; 132(6): 1477-1485, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30399106

RESUMO

OBJECTIVE: To evaluate whether C5b-9 concentrations in blood and urine are increased in preeclampsia with severe features. METHODS: The Complement and Preeclampsia in the Americas study is a prospective, multicenter case-control study performed at six centers in Colombia from November 2015 to July 2016. The case group included women with preeclampsia with severe features, and the control group included women who were healthy or had chronic hypertension, gestational hypertension, or preeclampsia without severe features. We enrolled two women in the control group for every woman in the case group. Soluble C5b-9 concentrations were measured by enzyme-linked immunosorbent assays in blood and urine. The primary outcome was C5b-9 concentrations in women in the case group compared with all women in the control group, and the secondary outcome was C5b-9 levels in women in the case group compared with individual control subgroups. Differences were assessed by test of medians, and associations were further evaluated by receiver operating characteristic curve analysis and logistic regression with α=0.05. RESULTS: Three hundred fifty-two patients were enrolled. Plasma C5b-9 concentrations did not differ significantly between women in the case group and those in the control group, but urine C5b-9 concentrations were higher in women in the case group (median [interquartile range] 9.9 [1.6-43.7] vs 1.8 [0.54-4.1] ng/mL, P<.001). In subgroup analysis, plasma C5b-9 concentrations were increased in women in the case group compared with healthy women in the control group (median [interquartile range] 2,778 [1,633-4,230] vs 1,374 [1,064-2,332] ng/mL, P<.001), and urine C5b-9 concentrations were increased in women in the case group compared with all control subgroups (P<.001). Using receiver operating characteristic analysis, urine C5b-9 concentrations differentiated preeclampsia with severe features from hypertensive women in the control group (area under the receiver operating characteristic curve 0.74, 95% CI 0.68-0.80). Urine C5b-9 22 ng/mL or greater (range 0-158.4 ng/mL) was the optimal cut point for diagnosis of preeclampsia with severe features with adjusted odds ratio of 10.0 (95% CI 3.5-28.8, P<.001). CONCLUSION: Urinary excretion of terminal complement effector C5b-9 is higher in women with preeclampsia with severe features compared with women with other hypertensive disorders of pregnancy and women without hypertension.


Assuntos
Complexo de Ataque à Membrana do Sistema Complemento/urina , Pré-Eclâmpsia/sangue , Pré-Eclâmpsia/urina , Adulto , Área Sob a Curva , Estudos de Casos e Controles , Ativação do Complemento , Complexo de Ataque à Membrana do Sistema Complemento/metabolismo , Feminino , Humanos , Hipertensão/sangue , Hipertensão/urina , Pré-Eclâmpsia/diagnóstico , Gravidez , Estudos Prospectivos , Curva ROC , Índice de Gravidade de Doença , Adulto Jovem
18.
J Immunol ; 201(11): 3184-3198, 2018 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-30404815

RESUMO

Sublytic C5b-9 formation on glomerular mesangial cells in rat Thy-1 nephritis (Thy-1N), a model of human mesangioproliferative glomerulonephritis, is accompanied by the production of proinflammatory cytokines, but the relationship between sublytic C5b-9 and cytokine synthesis and the underlying mechanism remains unclear. To explore the problems mentioned above, in this study, we first examined the levels of proinflammatory ILs (e.g., IL-23 and IL-36a) as well as transcription factor (KLF4) and coactivator (PCAF) in the renal tissues of Thy-1N rats and in the glomerular mesangial cell line (HBZY-1) stimulated by sublytic C5b-9. Then, we further determined the role of KLF4 and PCAF in sublytic C5b-9-induced IL-23 and IL-36a production as well as the related mechanism. Our results showed that the levels of KLF4, PCAF, IL-23, and IL-36a were obviously elevated. Mechanistic investigation revealed that sublytic C5b-9 stimulation could increase IL-23 and IL-36a synthesis through KLF4 and PCAF upregulation, and KLF4 and PCAF could form a complex, binding to the IL-23 or IL-36a promoter in a KLF4-dependent manner, causing gene transcription. Importantly, KLF4 acetylation by PCAF contributed to sublytic C5b-9-induced IL-23 and IL-36a transcription. Besides, the KLF4 binding regions on IL-23 or IL-36a promoters and the KLF4 lysine site acetylated by PCAF were identified. Furthermore, silencing renal KLF4 or PCAF gene could significantly inhibit IL-23 or IL-36a secretion and tissue damage of Thy-1N rats. Collectively, these findings implicate that the KLF4/PCAF interaction and KLF4 acetylation by PCAF play a pivotal role in the sublytic C5b-9-mediated IL-23 and IL-36a production of Thy-1N rats.


Assuntos
Complexo de Ataque à Membrana do Sistema Complemento/metabolismo , Interleucina-23/metabolismo , Interleucinas/metabolismo , Rim/metabolismo , Células Mesangiais/metabolismo , Nefrite/imunologia , Acetilação , Animais , Linhagem Celular , Humanos , Interleucina-23/genética , Interleucinas/genética , Rim/patologia , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Masculino , Células Mesangiais/patologia , Regiões Promotoras Genéticas/genética , RNA Interferente Pequeno/genética , Ratos , Ratos Sprague-Dawley , Antígenos Thy-1/metabolismo , Fatores de Transcrição de p300-CBP/genética , Fatores de Transcrição de p300-CBP/metabolismo
19.
J Immunol ; 201(12): 3717-3730, 2018 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-30429287

RESUMO

Complement activation is involved in the pathogenesis of ischemia reperfusion injury (IRI), which is an inevitable process during kidney transplantation. Therefore, complement-targeted therapeutics hold great potential in protecting the allografts from IRI. We observed universal deposition of C3d and membrane attack complex in human renal allografts with delayed graft function or biopsy-proved rejection, which confirmed the involvement of complement in IRI. Using FB-, C3-, C4-, C5-, C5aR1-, C5aR2-, and C6-deficient mice, we found that all components, except C5aR2 deficiency, significantly alleviated renal IRI to varying degrees. These gene deficiencies reduced local (deposition of C3d and membrane attack complex) and systemic (serum levels of C3a and C5a) complement activation, attenuated pathological damage, suppressed apoptosis, and restored the levels of multiple local cytokines (e.g., reduced IL-1ß, IL-9, and IL-12p40 and increased IL-4, IL-5, IL-10, and IL-13) in various gene-deficient mice, which resulted in the eventual recovery of renal function. In addition, we demonstrated that CRIg/FH, which is a targeted complement inhibitor for the classical and primarily alternative pathways, exerted a robust renoprotective effect that was comparable to gene deficiency using similar mechanisms. Further, we revealed that PI3K/AKT activation, predominantly in glomeruli that was remarkably inhibited by IRI, played an essential role in the CRIg/FH renoprotective effect. The specific PI3K antagonist duvelisib almost completely abrogated AKT phosphorylation, thus abolishing the renoprotective role of CRIg/FH. Our findings suggested that complement activation at multiple stages induced renal IRI, and CRIg/FH and/or PI3K/AKT agonists may hold the potential in ameliorating renal IRI.


Assuntos
Complemento C3d/metabolismo , Função Retardada do Enxerto/imunologia , Rejeição de Enxerto/imunologia , Transplante de Rim , Rim/patologia , Receptores de Complemento 3b/metabolismo , Traumatismo por Reperfusão/metabolismo , Animais , Células Cultivadas , Ativação do Complemento , Complemento C3d/genética , Complexo de Ataque à Membrana do Sistema Complemento/metabolismo , Citocinas/metabolismo , Humanos , Isoantígenos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais , Transplante Homólogo
20.
J Neuroinflammation ; 15(1): 294, 2018 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-30348195

RESUMO

BACKGROUND: Aquaporin-4-immunoglobulin G (AQP4-IgG) seropositive neuromyelitis optica spectrum disorder (herein called NMO) is an autoimmune disease of the central nervous system in which AQP4-IgG binding to AQP4 on astrocytes results in complement-dependent astrocyte injury and secondary inflammation, demyelination, and neuron loss. We previously reported evidence for a complement bystander mechanism for early oligodendrocyte injury in NMO. Herein, we tested the hypothesis that complement bystander injury, which involves diffusion to nearby cells of activated soluble complement components from complement-injured astrocytes, is a general phenomenon that may contribute to neuronal injury in NMO. METHODS: Primary cocultures of rat astrocytes and cortical neurons were established to study complement-dependent cell death after exposure to AQP4-IgG and complement. In animal experiments, AQP4-IgG was delivered to adult rats by intracerebral injection. Cell cultures and rat brain were studied by immunofluorescence. RESULTS: In primary astrocyte-neuron cocultures, addition of AQP4-IgG and complement resulted in death of neurons nearby astrocytes. Deposition of complement membrane attack complex C5b-9 was seen on neurons nearby astrocytes, whereas C1q, the initiating protein in the complement pathway, was seen only on astrocytes. Neuron death was not seen with a complement inhibitor, with C1q- or C6-depleted complement, in pure neuron cultures exposed to AQP4-IgG and complement or in cocultures exposed to an astrocyte toxin. Intracerebral injection in rats of AQP4-IgG and a fixable dead cell fluorescent marker produced death of neurons near astrocytes, with C5b-9 deposition. Neuron death was not seen in rats receiving a complement inhibitor or in AQP4-IgG-injected AQP4 knockout rats. CONCLUSION: These results support a novel mechanism for early neuron injury in NMO and provide evidence that complement bystander injury may be a general phenomenon for brain cell injury following AQP4-IgG-targeted astrocyte death.


Assuntos
Aquaporina 4/imunologia , Complemento C1q/metabolismo , Complexo de Ataque à Membrana do Sistema Complemento/metabolismo , Complexo de Ataque à Membrana do Sistema Complemento/toxicidade , Imunoglobulina G/sangue , Neuromielite Óptica/sangue , Neurônios/efeitos dos fármacos , Animais , Aquaporina 4/deficiência , Astrócitos/efeitos dos fármacos , Antígenos CD59/metabolismo , Células Cultivadas , Córtex Cerebral/citologia , Técnicas de Cocultura , Complemento C1q/toxicidade , Meios de Cultivo Condicionados/farmacologia , Modelos Animais de Doenças , Embrião de Mamíferos , Regulação da Expressão Gênica/efeitos dos fármacos , Imunoglobulina G/farmacologia , Proteínas do Tecido Nervoso/metabolismo , Neuromielite Óptica/induzido quimicamente , Ratos , Ratos Transgênicos , Fatores de Tempo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA