RESUMO
C2 is an attractive therapeutic target for many complement-mediated diseases. We developed Nab1B10, a new anti-C2 nanobody that potently and selectively inhibits both the classical and lectin pathways of complement activation. Mechanistically, Nab1B10 binds to the C2a portion of C2 and inhibits the assembly of C3 convertase C4b2a. Nab1B10 cross-reacts with monkey but not rodent C2 and inhibits classical pathway-mediated hemolysis. Using a new complement humanized mouse model of autoimmune hemolytic anemia (AIHA), we demonstrated that Nab1B10 abolished classical pathway complement activation-mediated hemolysis in vivo. We also developed C2-neutralizing bi- and tetra-valent antibodies based on Nab1B10 and found these antibodies significantly more potent than the other anti-C2 monoclonal antibody that is already in clinical trials. These data suggest that these novel C2-neutralizing nanobodies could be further developed as new therapeutics for many complement-mediated diseases, in which pathogenesis is dependent on the classical and/or lectin pathway of complement activation.
Assuntos
Anemia Hemolítica Autoimune , Complemento C2 , Camundongos , Animais , Complemento C2/metabolismo , Hemólise , Ativação do Complemento , Inativadores do ComplementoRESUMO
Several disorders associated with the total or partial absence of components of the human complement system are known. Deficiencies of classical pathway (CP) components are generally linked to systemic lupus erythematosus (SLE) or SLE-like syndromes. However, only approximately one-third of patients who lack C2 show mild symptoms of SLE. The relatively high frequency of homozygous C2 deficiency without or with minor disease manifestation suggests that there might be a compensatory mechanism which allows the activation of the CP of complement without the absolute requirement of C2. In this study we show that factor B (FB), the C2 homologue of the alternative pathway (AP) of complement, can substitute for C2. This was confirmed by using C4b as immobilised ligand and FB as analyte in Surface Plasmon Resonance (BIACORE). C2 binding to the immobilised C3b-like molecule C3(CH3NH2) was not seen. The estimated binding constant for C4bB complex formation was 2.00 * 10-5 [M]. We were further able to demonstrate that C4b supports the cleavage of Factor B by Factor D. Finally, cleavage of 125I-C3 by C4bBb was evaluated and gave strong evidence that the "hybrid" convertase C4bBb can cleave and activate C3 in vitro. Cleavage activity is very low, but consistent with some of the "C2-bypass" observations of others.
Assuntos
Complemento C4 , Lúpus Eritematoso Sistêmico , Ativação do Complemento , Complemento C2/metabolismo , Complemento C3/metabolismo , Convertases de Complemento C3-C5/metabolismo , Complemento C3b , Fator B do Complemento , Via Clássica do Complemento , HumanosRESUMO
Extremely rare reactions characterized by thrombosis and thrombocytopenia have been described in subjects that received ChAdOx1 nCoV-19 vaccination 5-16 days earlier. Although patients with vaccine-induced thrombotic thrombocytopenia (VITT) have high levels of antibodies to platelet factor 4 (PF4)-polyanion complexes, the exact mechanism of the development of thrombosis is still unknown. Here we reported serum studies as well as proteomics and genomics analyses demonstrating a massive complement activation potentially linked to the presence of anti-PF4 antibodies in a patient with severe VITT. At admission, complement activity of the classical and lectin pathways were absent (0% for both) with normal levels of the alternative pathway (73%) in association with elevated levels of the complement activation marker sC5b-9 (630 ng/mL [n.v. 139-462 ng/mL]) and anti-PF4 IgG (1.918 OD [n.v. 0.136-0.300 OD]). The immunoblotting analysis of C2 showed the complete disappearance of its normal band at 110 kDa. Intravenous immunoglobulin treatment allowed to recover complement activity of the classical pathway (91%) and lectin pathway (115%), to reduce levels of sC5b-9 (135 ng/mL) and anti-PF4 IgG (0.681 OD) and to normalize the C2 pattern at immunoblotting. Proteomics and genomics analyses in addition to serum studies showed that the absence of complement activity during VITT was not linked to alterations of the C2 gene but rather to a strong complement activation leading to C2 consumption. Our data in a single patient suggest monitoring complement parameters in other VITT patients considering also the possibility to target complement activation with specific drugs.
Assuntos
Vacinas contra COVID-19/efeitos adversos , COVID-19/prevenção & controle , Complemento C2 , Complexo de Ataque à Membrana do Sistema Complemento , Via Clássica do Complemento , Lectina de Ligação a Manose da Via do Complemento , Púrpura Trombocitopênica Trombótica , SARS-CoV-2 , Adulto , Autoanticorpos/sangue , Vacinas contra COVID-19/administração & dosagem , ChAdOx1 nCoV-19 , Complemento C2/genética , Complemento C2/metabolismo , Complexo de Ataque à Membrana do Sistema Complemento/genética , Complexo de Ataque à Membrana do Sistema Complemento/metabolismo , Via Clássica do Complemento/efeitos dos fármacos , Via Clássica do Complemento/genética , Lectina de Ligação a Manose da Via do Complemento/efeitos dos fármacos , Lectina de Ligação a Manose da Via do Complemento/genética , Feminino , Humanos , Fator Plaquetário 4/sangue , Púrpura Trombocitopênica Trombótica/sangue , Púrpura Trombocitopênica Trombótica/induzido quimicamente , Púrpura Trombocitopênica Trombótica/genéticaRESUMO
BACKGROUND: Activation of the classical and lectin pathway of complement may contribute to tissue damage and organ dysfunction of antibody-mediated diseases and ischemia-reperfusion conditions. Complement factors are being considered as targets for therapeutic intervention. OBJECTIVE: We sought to characterize ARGX-117, a humanized inhibitory monoclonal antibody against complement C2. METHODS: The mode-of-action and binding characteristics of ARGX-117 were investigated in detail. Furthermore, its efficacy was analyzed in in vitro complement cytotoxicity assays. Finally, a pharmacokinetic/pharmacodynamic study was conducted in cynomolgus monkeys. RESULTS: Through binding to the Sushi-2 domain of C2, ARGX-117 prevents the formation of the C3 proconvertase and inhibits classical and lectin pathway activation upstream of C3 activation. As ARGX-117 does not inhibit the alternative pathway, it is expected not to affect the antimicrobial activity of this complement pathway. ARGX-117 prevents complement-mediated cytotoxicity in in vitro models for autoimmune hemolytic anemia and antibody-mediated rejection of organ transplants. ARGX-117 exhibits pH- and calcium-dependent target binding and is Fc-engineered to increase affinity at acidic pH to the neonatal Fc receptor, and to reduce effector functions. In cynomolgus monkeys, ARGX-117 dose-dependently reduces free C2 levels and classical pathway activity. A 2-dose regimen of 80 and 20 mg/kg separated by a week, resulted in profound reduction of classical pathway activity lasting for at least 7 weeks. CONCLUSIONS: ARGX-117 is a promising new complement inhibitor that is uniquely positioned to target both the classical and lectin pathways while leaving the alternative pathway intact.
Assuntos
Anticorpos Monoclonais/farmacologia , Complemento C2/antagonistas & inibidores , Inativadores do Complemento/farmacologia , Animais , Anticorpos Monoclonais/sangue , Anticorpos Monoclonais/farmacocinética , Cálcio , Ativação do Complemento/efeitos dos fármacos , Complemento C2/análise , Complemento C2/metabolismo , Inativadores do Complemento/sangue , Inativadores do Complemento/farmacocinética , Mapeamento de Epitopos , Feminino , Humanos , Concentração de Íons de Hidrogênio , Macaca fascicularis , MasculinoRESUMO
Bone marrow-derived mast cells (BMMCs) are often used as a model system for studies of the role of MCs in health and disease. These cells are relatively easy to obtain from total bone marrow cells by culturing under the influence of IL-3 or stem cell factor (SCF). After 3 to 4 weeks in culture, a nearly homogenous cell population of toluidine blue-positive cells are often obtained. However, the question is how relevant equivalents these cells are to normal tissue MCs. By comparing the total transcriptome of purified peritoneal MCs with BMMCs, here we obtained a comparative view of these cells. We found several important transcripts that were expressed at very high levels in peritoneal MCs, but were almost totally absent from the BMMCs, including the major chymotryptic granule protease Mcpt4, the neurotrophin receptor Gfra2, the substance P receptor Mrgprb2, the metalloprotease Adamts9 and the complement factor 2 (C2). In addition, there were a number of other molecules that were expressed at much higher levels in peritoneal MCs than in BMMCs, including the transcription factors Myb and Meis2, the MilR1 (Allergin), Hdc (Histidine decarboxylase), Tarm1 and the IL-3 receptor alpha chain. We also found many transcripts that were highly expressed in BMMCs but were absent or expressed at low levels in the peritoneal MCs. However, there were also numerous MC-related transcripts that were expressed at similar levels in the two populations of cells, but almost absent in peritoneal macrophages and B cells. These results reveal that the transcriptome of BMMCs shows many similarities, but also many differences to that of tissue MCs. BMMCs can thereby serve as suitable models in many settings concerning the biology of MCs, but our findings also emphasize that great care should be taken when extrapolating findings from BMMCs to the in vivo function of tissue-resident MCs.
Assuntos
Linfócitos B/metabolismo , Células da Medula Óssea/metabolismo , Macrófagos/metabolismo , Mastócitos/metabolismo , Peritônio/metabolismo , Transcriptoma , Proteína ADAMTS9/genética , Proteína ADAMTS9/metabolismo , Animais , Linfócitos B/citologia , Biomarcadores/metabolismo , Células da Medula Óssea/citologia , Complemento C2/genética , Complemento C2/metabolismo , Feminino , Regulação da Expressão Gênica , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Histidina Descarboxilase/genética , Histidina Descarboxilase/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Macrófagos/citologia , Mastócitos/citologia , Camundongos , Camundongos Endogâmicos BALB C , Especificidade de Órgãos , Peritônio/citologia , Proteínas Proto-Oncogênicas c-myb/genética , Proteínas Proto-Oncogênicas c-myb/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismo , Receptores de Interleucina-3/genética , Receptores de Interleucina-3/metabolismo , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismoRESUMO
BACKGROUND: Single nucleotide polymorphism (SNP) of complement component 2 (C2) has been found to be significantly associated with hepatocellular carcinoma (HCC). However, little is known about the role and mechanism of C2 in HCC. In the present study, we aimed to explore the prognostic value of C2 and its correlation with tumor-infiltrating immune cells in HCC. MATERIALS AND METHODS: mRNA expression was downloaded from TCGA (365 HCC patients and 50 healthy controls), GSE14520 (220 HCC patients and 220 adjacent normal tissues), and ICGC HCC (232 HCC patients) cohorts. Unpaired Student's t-tests or ANOVA tests were used to evaluate differences of C2 expression. Univariate and multivariate analyses were used to analyze the prognostic value of C2. CIBERSORT was used to calculate the proportion of 22 kinds of tumor-infiltrating immune cells. RESULTS: Significantly lower C2 expression was found at HCC compared to healthy controls, and C2 was associated with TNM stages. Higher C2 expression was significantly associated with better prognosis, and multivariate analysis showed that C2 was also an independent factor for the prognosis of HCC. Moreover, elevated CD4 T cells were found at HCC patients with higher C2 expression while the higher proportion of macrophage M0 cells was found in HCC patients with lower C2 expression. KEGG analysis showed that "cell cycle," "AMPK signaling pathway," and "PPAR signaling pathway" were enriched in HCC patients with higher C2 expression. CONCLUSION: C2 is a prognostic factor for HCC and may be used as a therapeutic target for future treatment of HCC.
Assuntos
Carcinoma Hepatocelular , Complemento C2 , Neoplasias Hepáticas , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma Hepatocelular/diagnóstico , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/mortalidade , Carcinoma Hepatocelular/patologia , Complemento C2/genética , Complemento C2/metabolismo , Bases de Dados Genéticas , Feminino , Humanos , Neoplasias Hepáticas/diagnóstico , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/mortalidade , Neoplasias Hepáticas/patologia , Masculino , Pessoa de Meia-Idade , Prognóstico , Transdução de Sinais/genética , Transcriptoma/genética , Adulto JovemRESUMO
Complement factor B/C2 family (Bf/C2F) proteins are core complement system components in vertebrates that are absent in invertebrates and have been lost by numerous species, raising evolutionary questions. At least 3 duplication events have occurred from Cnidaria (ancestor) to mammals. Type II Bf/C2 genes appeared during separation of Proterostomia and Deuterostomes. The second event occurred during separation of vertebrates and invertebrates, yielding type II-2 Bf/C2. The third event occurred when jawed and jawless fish were separated, eventually producing Bf and C2 genes. Herein, we report the second mollusc Sinonovacula constricta Bf/C2-type gene (ScBf). ScBf is similar to Ruditapes decussatus Bf-like because both lack the first complement control protein module at the N terminus present in mammalian Bf/C2 proteins. Uniquely, the Ser protease (SP) module at the C terminus of ScBf is â¼50 aa longer than in other complement factor B/C2-type (Bf/C2T) proteins, and is Glu-rich. Bf/C2T proteins in molluscs lack the catalytic Ser in the SP module. Surprisingly, ScBf regulates rabbit erythrocyte agglutination, during which it is localized on the erythrocyte surface. Thus, ScBf may mediate the agglutination cascade and may be an upstream regulator of this process. Our findings provide new insight into the origin of the Bf/C2F.-Peng, M., Li, Z., Niu, D., Liu, X., Dong, Z., Li, J. Complement factor B/C2 in molluscs regulates agglutination and illuminates evolution of the Bf/C2 family.
Assuntos
Infecções Bacterianas/veterinária , Complemento C2/metabolismo , Fator B do Complemento/metabolismo , Eritrócitos/patologia , Evolução Molecular , Doenças dos Peixes/patologia , Aglutinação , Animais , Bactérias/crescimento & desenvolvimento , Infecções Bacterianas/genética , Infecções Bacterianas/metabolismo , Infecções Bacterianas/patologia , Complemento C2/genética , Fator B do Complemento/genética , Eritrócitos/metabolismo , Eritrócitos/microbiologia , Doenças dos Peixes/genética , Doenças dos Peixes/metabolismo , Moluscos , Filogenia , CoelhosRESUMO
Macrophage-based immune dysregulation plays a critical role in development of delayed gastric emptying in diabetic mice. Loss of anti-inflammatory macrophages and increased expression of genes associated with pro-inflammatory macrophages has been reported in full-thickness gastric biopsies from gastroparesis patients. We aimed to determine broader protein expression (proteomics) and protein-based signaling pathways in gastric biopsies of diabetic (DG) and idiopathic gastroparesis (IG) patients. Additionally, we determined correlations between protein expressions, gastric emptying, and symptoms. Full-thickness gastric antrum biopsies were obtained from nine DG patients, seven IG patients, and five nondiabetic controls. Aptamer-based SomaLogic tissue scan that quantitatively identifies 1,305 human proteins was used. Protein fold changes were computed, and differential expressions were calculated using Limma. Ingenuity pathway analysis and correlations were carried out. Multiple-testing corrected P < 0.05 was considered statistically significant. Seventy-three proteins were differentially expressed in DG, 132 proteins were differentially expressed in IG, and 40 proteins were common to DG and IG. In both DG and IG, "Role of Macrophages, Fibroblasts and Endothelial Cells" was the most statistically significant altered pathway [DG false discovery rate (FDR) = 7.9 × 10-9; IG FDR = 6.3 × 10-12]. In DG, properdin expression correlated with GCSI bloating (r = -0.99, FDR = 0.02) and expressions of prostaglandin G/H synthase 2, protein kinase C-ζ type, and complement C2 correlated with 4 h gastric retention (r = -0.97, FDR = 0.03 for all). No correlations were found between proteins and symptoms or gastric emptying in IG. Protein expression changes suggest a central role of macrophage-driven immune dysregulation in gastroparesis, specifically, complement activation in diabetic gastroparesis.NEW & NOTEWORTHY This study uses SOMAscan, a novel proteomics assay for determination of altered proteins and associated molecular pathways in human gastroparesis. Seventy-three proteins were changed in diabetic gastroparesis, 132 in idiopathic gastroparesis compared with controls. Forty proteins were common in both. Macrophage-based immune dysregulation pathway was most significantly affected in both diabetic and idiopathic gastroparesis. Proteins involved in the complement and prostaglandin synthesis pathway were associated with symptoms and gastric emptying delay in diabetic gastroparesis.
Assuntos
Complicações do Diabetes/genética , Gastroparesia/genética , Proteoma/genética , Adulto , Idoso , Complemento C2/genética , Complemento C2/metabolismo , Complicações do Diabetes/metabolismo , Complicações do Diabetes/fisiopatologia , Células Endoteliais/metabolismo , Feminino , Fibroblastos/metabolismo , Esvaziamento Gástrico , Gastroparesia/etiologia , Gastroparesia/metabolismo , Gastroparesia/fisiopatologia , Humanos , Macrófagos/metabolismo , Masculino , Pessoa de Meia-Idade , Prostaglandina-Endoperóxido Sintases/genética , Prostaglandina-Endoperóxido Sintases/metabolismo , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Proteoma/metabolismoRESUMO
BACKGROUND: Schistosomiasis remains an important global public health problem, as millions of people are at risk of acquiring infection. An ideal method for sustainable control of schistosomiasis would be to develop an efficient vaccine. Schistosomes can survive in the host vascular system by immune evasion, regulating the host complement cascade. Schistosoma japonicum tetraspanning orphan receptor (SjTOR) is a complement regulator, which is a tegument membrane protein. To date there is no experimental evidence to explain the function of SjTOR. RESULTS: We cloned the first extracellular domain of the SjTOR (SjTOR-ed1) gene and expressed the gene in Escherichia coli. The expression level of SjTOR in different developmental stages of S. japonicum was assessed by quantitative real-time RT-PCR. Western blotting showed that recombinant SjTOR-ed1 (rSjTOR-ed1) could be recognised by schistosome-infected mouse serum. Immunolocalization indicated that the protein was mainly distributed on the tegument of the parasite. Haemolytic assays and ELISA revealed that rSjTOR-ed1 could inhibit complement hemolysis and bind to complement C2. Purified rSjTOR-ed1 emulsified with ISA206 adjuvant could induce a significant reduction of worm burden from 24.51 to 26.51%, and liver egg numbers from 32.92 to 39.62% in two independent trials in mice. CONCLUSIONS: The results of this study indicated that rSjTOR-ed1 could inhibit complement hemolysis and bind to complement C2, and it is a potential vaccine candidate that protects against S. japonicum infection.
Assuntos
Complemento C2/metabolismo , Proteínas de Helminto/metabolismo , Interações Hospedeiro-Patógeno , Fatores Imunológicos/metabolismo , Ribonucleases/metabolismo , Schistosoma japonicum/imunologia , Esquistossomose/imunologia , Animais , Modelos Animais de Doenças , Proteínas de Helminto/genética , Camundongos Endogâmicos C57BL , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ribonucleases/genética , Células Th2/imunologiaRESUMO
All 3 activation pathways of complement-the classic pathway (CP), the alternative pathway, and the lectin pathway (LP)- converge into a common central event: the cleavage and activation of the abundant third complement component, C3, via formation of C3-activating enzymes (C3 convertases). The fourth complement component, C4, and the second component, C2, are indispensable constituents of the C3 convertase complex, C4bC2a, which is formed by both the CP and the LP. Whereas in the absence of C4, CP can no longer activate C3, LP retains a residual but physiologically critical capacity to convert native C3 into its activation fragments, C3a and C3b. This residual C4 and/or C2 bypass route is dependent on LP-specific mannan-binding lectin-associated serine protease-2. By using various serum sources with defined complement deficiencies, we demonstrate that, under physiologic conditions LP-specific C4 and/or C2 bypass activation of C3 is mediated by direct cleavage of native C3 by mannan-binding lectin-associated serine protease-2 bound to LP-activation complexes captured on ligand-coated surfaces.-Yaseen, S., Demopulos, G., Dudler, T., Yabuki, M., Wood, C. L., Cummings, W. J., Tjoelker, L. W., Fujita, T., Sacks, S., Garred, P., Andrew, P., Sim, R. B., Lachmann, P. J., Wallis, R., Lynch, N., Schwaeble, W. J. Lectin pathway effector enzyme mannan-binding lectin-associated serine protease-2 can activate native complement C3 in absence of C4 and/or C2.
Assuntos
Ativação do Complemento/fisiologia , Complemento C2/metabolismo , Complemento C3/metabolismo , Complemento C4/metabolismo , Lectinas/metabolismo , Serina Proteases Associadas a Proteína de Ligação a Manose/metabolismo , HumanosRESUMO
The complement system plays a key role in innate immunity, inflammation, and coagulation. The system is delicately balanced by negative regulatory mechanisms that modulate the host response to pathogen invasion and injury. The serpin, C1-esterase inhibitor (C1-INH), is the only known plasma inhibitor of C1s, the initiating serine protease of the classical pathway of complement. Like other serpin-protease partners, C1-INH interaction with C1s is accelerated by polyanions such as heparin. Polyphosphate (polyP) is a naturally occurring polyanion with effects on coagulation and complement. We recently found that polyP binds to C1-INH, prompting us to consider whether polyP acts as a cofactor for C1-INH interactions with its target proteases. We show that polyP dampens C1s-mediated activation of the classical pathway in a polymer length- and concentration-dependent manner by accelerating C1-INH neutralization of C1s cleavage of C4 and C2. PolyP significantly increases the rate of interaction between C1s and C1-INH, to an extent comparable to heparin, with an exosite on the serine protease domain of the enzyme playing a major role in this interaction. In a serum-based cell culture system, polyP significantly suppressed C4d deposition on endothelial cells, generated via the classical and lectin pathways. Moreover, polyP and C1-INH colocalize in activated platelets, suggesting that their interactions are physiologically relevant. In summary, like heparin, polyP is a naturally occurring cofactor for the C1s:C1-INH interaction and thus an important regulator of complement activation. The findings may provide novel insights into mechanisms underlying inflammatory diseases and the development of new therapies.
Assuntos
Proteínas Inativadoras do Complemento 1/metabolismo , Proteínas do Sistema Complemento/metabolismo , Polifosfatos/metabolismo , Sítios de Ligação , Plaquetas/imunologia , Plaquetas/metabolismo , Células Cultivadas , Proteína Inibidora do Complemento C1 , Complemento C1s/química , Complemento C1s/metabolismo , Complemento C2/metabolismo , Complemento C4/metabolismo , Via Clássica do Complemento , Células Endoteliais/imunologia , Células Endoteliais/metabolismo , Heparina/metabolismo , Humanos , Técnicas In Vitro , Polifosfatos/químicaRESUMO
Parasitic nematodes manufacture various carbohydrate-linked phosphorylcholine (PCh)-containing molecules, including ES-62, a protein with an N-linked glycan terminally substituted with PCh. The PCh component is biologically important because it is required for immunomodulatory effects. We showed that most ES-62 was bound to a single protein, C-reactive protein (CRP), in normal human serum, displaying a calcium-dependent, high-avidity interaction and ability to form large complexes. Unexpectedly, CRP binding to ES-62 failed to efficiently activate complement as far as the C3 convertase stage in comparison with PCh-BSA and PCh-containing Streptococcus pneumoniae cell wall polysaccharide. C1q capture assays demonstrated an ES-62-CRP-C1q interaction in serum. The three ligands all activated C1 and generated C4b to similar extents. However, a C2a active site was not generated following ES-62 binding to CRP, demonstrating that C2 cleavage was far less efficient for ES-62-containing complexes. We proposed that failure of C2 cleavage was due to the flexible nature of carbohydrate-bound PCh and that reduced proximity of the C1 complex was the reason that C2 was poorly cleaved. This was confirmed using synthetic analogues that were similar to ES-62 only in respect of having a flexible PCh. Furthermore, ES-62 was shown to deplete early complement components, such as the rate-limiting C4, following CRP interaction and thereby inhibit classical pathway activation. Thus, flexible PCh-glycan represents a novel mechanism for subversion of complement activation. These data illustrate the importance of the rate-limiting C4/C2 stage of complement activation and reveal a new addition to the repertoire of ES-62 immunomodulatory mechanisms with possible therapeutic applications.
Assuntos
Configuração de Carboidratos , Ativação do Complemento/efeitos dos fármacos , Complemento C2/metabolismo , Complemento C4/metabolismo , Via Clássica do Complemento/efeitos dos fármacos , Proteínas de Helminto/farmacologia , Fosforilcolina/metabolismo , Sítios de Ligação , Proteína C-Reativa/metabolismo , Convertases de Complemento C3-C5/metabolismo , Humanos , Ressonância de Plasmônio de SuperfícieRESUMO
BACKGROUND: There is growing evidence of the importance of nutrition in age-related macular degeneration (AMD), but few studies have explored associations with folate and B vitamins. No effective therapeutic strategy for geographic atrophy (GA) is available, and prevention could be of great value. OBJECTIVE: We investigated associations between dietary folate, B vitamins, and progression to GA and whether these associations might be modified by genetic susceptibility. DESIGN: Among 2525 subjects (4663 eyes) in the Age-Related Eye Disease Study, 405 subjects (528 eyes) progressed to GA over 13 y. Folate and B vitamins were log transformed and calorie adjusted separately for men and women. Ten loci in 7 AMD genes [complement factor H, age-related maculopathy susceptibility 2/high-temperature requirement A serine peptidase 1, complement component 2, complement component 3, complement factor B, collagen type VIII α 1, and RAD51 paralog B] were examined. Survival analysis was used to assess associations between incident GA and dietary intake of folate and B vitamins. Interaction effects between these nutrients and genetic variation on AMD risk were also evaluated. Subjects with at least one eye free of advanced AMD at baseline were included in these analyses. RESULTS: There was a reduced risk of progression to GA with increasing intake of thiamin, riboflavin, and folate after adjusting for age, sex, and total energy intake (P-trend = 0.01, 0.03, and 0.001, respectively). After adjustment for demographic, behavioral, ocular, and genetic covariates, trends remained statistically significant for folate (P-trend = 0.007) and were borderline for thiamin (P-trend = 0.05). Riboflavin did not retain statistical significance (P-trend = 0.20). Folate was significantly associated with lower risk of incident GA among subjects homozygous for the complement component 3 (C3) R102G rs2230199 nonrisk genotype (CC) (HR = 0.43; 95% CI: 0.27, 0.70; P = 0.0005) but not subjects carrying the risk allele (G) (P = 0.76). Neither folate nor any B vitamin was significantly associated with neovascular AMD. CONCLUSIONS: High folate intake was associated with a reduced risk of progression to GA. This relation could be modified by genetic susceptibility, particularly related to the C3 genotype. This trial was registered at clinicaltrials.gov as NCT00594672.
Assuntos
Ácido Fólico/administração & dosagem , Predisposição Genética para Doença , Atrofia Geográfica/genética , Degeneração Macular/genética , Complexo Vitamínico B/administração & dosagem , Idoso , Índice de Massa Corporal , Colágeno Tipo VIII/genética , Colágeno Tipo VIII/metabolismo , Complemento C2/genética , Complemento C2/metabolismo , Complemento C3/genética , Complemento C3/metabolismo , Fator B do Complemento/genética , Fator B do Complemento/metabolismo , Fator H do Complemento/genética , Fator H do Complemento/metabolismo , Progressão da Doença , Feminino , Serina Peptidase 1 de Requerimento de Alta Temperatura A , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Proteínas/genética , Proteínas/metabolismo , Riboflavina/administração & dosagem , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Tiamina/administração & dosagemRESUMO
The article considers the developed techniques of detection of functional activity of components C1, C2, C3, C4 and membrane attacking complex of classical pathway and also factors B and D of alternative pathway of human complement using automated device measurement of immobilizing effect on infusorians Tetrahymena pyriformis. The techniques are based on application of artificially produced reagents containing all necessary components of compliment besides testing one. The mathematical mode of activities calculation is developed. The linear dependency of velocity of infusorians immobilizing from functional activities of testing component. The relevance of techniques is demonstrated by correlation of results derived using the proposed mode with the developed earlier hemolytic mode.
Assuntos
Eritrócitos/metabolismo , Hemólise , Tetrahymena pyriformis/crescimento & desenvolvimento , Complemento C1/metabolismo , Complemento C2/metabolismo , Complemento C3/metabolismo , Complemento C4/metabolismo , Humanos , Tetrahymena pyriformis/metabolismoRESUMO
The pathogenic bacterium Staphylococcus aureus actively evades many aspects of human innate immunity by expressing a series of small inhibitory proteins. A number of these proteins inhibit the complement system, which labels bacteria for phagocytosis and generates inflammatory chemoattractants. Although the majority of staphylococcal complement inhibitors act on the alternative pathway to block the amplification loop, only a few proteins act on the initial recognition cascades that constitute the classical pathway (CP) and lectin pathway (LP). We screened a collection of recombinant, secreted staphylococcal proteins to determine whether S. aureus produces other molecules that inhibit the CP and/or LP. Using this approach, we identified the extracellular adherence protein (Eap) as a potent, specific inhibitor of both the CP and LP. We found that Eap blocked CP/LP-dependent activation of C3, but not C4, and that Eap likewise inhibited deposition of C3b on the surface of S. aureus cells. In turn, this significantly diminished the extent of S. aureus opsonophagocytosis and killing by neutrophils. This combination of functional properties suggested that Eap acts specifically at the level of the CP/LP C3 convertase (C4b2a). Indeed, we demonstrated a direct, nanomolar-affinity interaction of Eap with C4b. Eap binding to C4b inhibited binding of both full-length C2 and its C2b fragment, which indicated that Eap disrupts formation of the CP/LP C3 proconvertase (C4b2). As a whole, our results demonstrate that S. aureus inhibits two initiation routes of complement by expression of the Eap protein, and thereby define a novel mechanism of immune evasion.
Assuntos
Proteínas de Bactérias/imunologia , Proteínas de Bactérias/metabolismo , C3 Convertase da Via Alternativa do Complemento/antagonistas & inibidores , Via Clássica do Complemento/imunologia , Lectina de Ligação a Manose da Via do Complemento/imunologia , Proteínas de Ligação a RNA/imunologia , Proteínas de Ligação a RNA/metabolismo , Staphylococcus aureus/imunologia , Staphylococcus aureus/metabolismo , Proteínas de Bactérias/química , Sítios de Ligação , Complemento C2/imunologia , Complemento C2/metabolismo , Complemento C3b/imunologia , Complemento C3b/metabolismo , Complemento C4b/imunologia , Complemento C4b/metabolismo , Citotoxicidade Imunológica , Humanos , Modelos Imunológicos , Neutrófilos/imunologia , Fagocitose/imunologia , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas de Ligação a RNA/química , Infecções Estafilocócicas/imunologia , Infecções Estafilocócicas/metabolismoRESUMO
The complement system plays an important role in both innate and adaptive host defense against the invading microorganisms in vertebrates. It can be activated by three pathways: the classical, alternative and lectin pathways. Bf/C2 and C4, as members of complement, play a pivotal role in the activation of the complement system. In our study, we identified Bf/C2 and C4 genes and genomic structure in miiuy croaker, and expression patterns of Bf/C2 and C4 genes was analyzed. In healthy miiuy croaker tissues, Bf/C2 and C4 genes were found to be ubiquitously expressed in all ten tested tissues. Analysis of expression of Bf/C2 and C4 genes after bacterial infection showed a significant up-regulated in liver. The evolutionary analysis showed that the ancestral lineages of Bf/C2 and C4 genes in mammals and fishes experienced positive selection indicated that the ancestors of mammals and fishes had further evolved to adapt to their environment, respectively. A series of maximum likelihood (ML) methods were used to study the evolution on vertebrates' Bf/C2 and C4 genes. One and five positive selection sites were found in mammals of Bf/C2 and C4 genes, but no positive selection site was found in fishes of Bf/C2 and C4 genes, indicating that Bf/C2 and C4 genes in mammals and fishes underwent different evolutionary patterns.
Assuntos
Infecções Bacterianas/veterinária , Complemento C2/metabolismo , Complemento C4/metabolismo , Evolução Molecular , Doenças dos Peixes/genética , Doenças dos Peixes/imunologia , Modelos Moleculares , Perciformes , Sequência de Aminoácidos , Animais , Infecções Bacterianas/genética , Infecções Bacterianas/imunologia , Infecções Bacterianas/metabolismo , Sequência de Bases , Teorema de Bayes , Complemento C2/genética , Complemento C4/genética , Primers do DNA/genética , Doenças dos Peixes/metabolismo , Funções Verossimilhança , Fígado/metabolismo , Modelos Genéticos , Dados de Sequência Molecular , Filogenia , Conformação Proteica , Seleção Genética , Análise de Sequência de DNARESUMO
BACKGROUND: We previously reported on subtypes of polypoidal choroidal vasculopathy (PCV), and categorized PCV as polypoidal choroidal neovascularization (CNV) and typical PCV. The aim of this study was to clarify whether complement component 2 (C2) and complement factor B (CFB) genotypes are associated with subtypes of polypoidal choroidal vasculopathy, such as polypoidal CNV and typical PCV. METHODS: First, we categorized 677 patients into typical age-related macular degeneration (tAMD; 250 patients), PCV (376) and retinal angiomatous proliferation (RAP; 51). Second, we categorized 282 patients with PCV as having polypoidal CNV (84 patients) or typical PCV (198) based on indocyanine green angiographic findings. In total, 274 subjects without AMD, such as PCV and CNV, served as controls. A SNP (rs547154) in the C2 gene and three SNPs (rs541862, rs2072633, rs4151667) in the CFB gene were genotyped, and case-control studies were performed in subjects with these PCV subtypes. RESULTS: In tAMD, no SNPs were associated with allele distributions. In PCV, rs547154 and rs2072633 were associated with allele distributions. RAP was only associated with rs2072633. After logistic regression analysis with adjustment for confounding factors, tAMD, PCV and RAP were found to be associated with rs2072633.As to PCV subtypes, there were significant differences in the distributions of rs547154, rs541862 and rs2072633 in the case-control studies for polypoidal CNV, but not between the typical PCV and control groups. Logistic regression analysis with adjustment for confounding factors showed the distributions of rs547154, rs541862 and rs2072633 to differ significantly between the controls and polypoidal CNV cases and that these SNPs were protective. The A/A genotype of rs2072633 was significantly more common in the polypoidal CNV than in the typical PCV group (p = 0.03), even with adjustment for polyp number and greatest linear dimension. CONCLUSIONS: PCV might be genetically divisible into polypoidal CNV and typical PCV. The C2 and CFB gene variants were shown to be associated with polypoidal CNV. Typical PCV was not associated with variants in these genes.
Assuntos
Corioide/patologia , Neovascularização de Coroide/genética , Complemento C2/genética , Fator B do Complemento/genética , Predisposição Genética para Doença , Idoso , Idoso de 80 Anos ou mais , Corioide/metabolismo , Neovascularização de Coroide/diagnóstico , Neovascularização de Coroide/metabolismo , Complemento C2/metabolismo , Fator B do Complemento/metabolismo , Feminino , Angiofluoresceinografia , Seguimentos , Fundo de Olho , Genótipo , Humanos , Masculino , Estudos RetrospectivosRESUMO
PURPOSE: We assessed the association between complement pathway genes and age-related macular degeneration (AMD) in a Chinese population. METHODS: In a case-control study, 165 AMD patients and 216 unrelated controls were recruited from two hospitals in central China. We selected and genotyped six single nucleotide polymorphisms (SNPs) of four complement pathway genes, including rs800292 and rs1410996 of complement H (CFH), rs9332739 of complement 2 (C2), rs4151667 of complement factor B (CFB), and rs2241394 and rs2230199 of complement 3 (C3). The associations between SNPs and AMD, adjusted by age and sex, were assessed by using logistic regression models and haplotype association analysis. RESULTS: In our study, two SNPs of CFH and their haplotypes were associated significantly with AMD, and the adjusted odd ratios (ORs) were 2.45 (95% confidence interval [CI] 1.25-4.79) for rs800292 (genotype GG versus AA), 2.49 (95% CI 1.24-5.00) for rs1410996 (genotype TT versus CC), and 4.45 (95% CI 2.32-8.55) for haplotype block of rs800292-rs1410996 (haplotype G-C versus A-C), respectively. The haplotype of C2/CFB also was associated significantly with AMD, and the adjusted OR was 8.86 (95% CI 1.88-41.69) for the haplotype block of rs9332739-rs4151667 (haplotype G-A versus G-T), though no relationship was found in genotype association analysis of the two SNPs of C2/CFB. With the sample size of our study, no relationship was found for AMD and the two SNPs of C3. CONCLUSIONS: Gene variants in CFH and C2/CFB contribute to AMD in the Chinese population.
Assuntos
Povo Asiático/genética , Povo Asiático/estatística & dados numéricos , Proteínas do Sistema Complemento/genética , Degeneração Macular/etnologia , Degeneração Macular/genética , Polimorfismo de Nucleotídeo Único/genética , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , China/epidemiologia , Complemento C2/genética , Complemento C2/metabolismo , Complemento C3/genética , Complemento C3/metabolismo , Fator B do Complemento/genética , Fator B do Complemento/metabolismo , Fator H do Complemento/genética , Fator H do Complemento/metabolismo , Proteínas do Sistema Complemento/metabolismo , Feminino , Predisposição Genética para Doença/etnologia , Predisposição Genética para Doença/genética , Haplótipos , Humanos , Degeneração Macular/metabolismo , Masculino , Pessoa de Meia-IdadeRESUMO
PURPOSE: Association between genetic variants in complement factor H (CFH), factor B (CFB), component 2 (C2), and in the ARMS2/HTRA1 region with age-related macular degeneration (AMD) comes mainly from studies of European ancestry and case-control studies of late-stage disease. We investigated associations of both early and late AMD with these variants in a population-based study of people aged 60 years and older in India. METHODS: Fundus images were graded using the Wisconsin Age-Related Maculopathy Grading System and participants assigned to one of four mutually exclusive stages based on the worse affected eye (0 = no AMD, 1-3 = early AMD, 4 = late AMD). Multinomial logistic regression was used to derive risk ratios (RR) accounting for sampling method and adjusting for age, sex, and study center. RESULTS: Of 3569 participants, 53.2% had no signs of amd, 45.6% had features of early amd, and 1.2% had late amd. CFH (RS1061170), C2 (RS547154), OR CFB (RS438999) was not associated with early or late AMD. In the ARMS2 locus, RS10490924 was associated with both early (adjusted RR 1.22, 95% confidence interval [CI]: 1.13-1.33, P < 0.0001) and late AMD (adjusted RR 1.81, 95% CI: 1.15-2.86; P = 0.01); rs2672598 was associated only with early AMD (adjusted RR 1.12, 95% CI: 1.02-1.23; P = 0.02); rs10490923 was not associated with early or late AMD. CONCLUSIONS: Two variants in ARMS2/HTRA1 were associated with increased risk of early AMD, and for one of these, the increased risk was also evident for late AMD. The study provides new insights into the role of these variants in early stages of AMD in India.
Assuntos
Complemento C2/genética , DNA/genética , Degeneração Macular/genética , Polimorfismo Genético , Proteínas/genética , Serina Endopeptidases/genética , Idoso , Complemento C2/metabolismo , Feminino , Frequência do Gene , Predisposição Genética para Doença , Genótipo , Serina Peptidase 1 de Requerimento de Alta Temperatura A , Humanos , Índia/epidemiologia , Degeneração Macular/epidemiologia , Degeneração Macular/metabolismo , Masculino , Pessoa de Meia-Idade , Prevalência , Proteínas/metabolismo , Serina Endopeptidases/metabolismoRESUMO
Zearalenone (hereafter referred to as ZEA) is a nonsteroidal estrogenic mycotoxin produced by several Fusarium spp. on cereal grains. ZEA is one of the most hazardous natural endocrine disrupting chemicals (EDC) which induces hyper estrogenic responses in mammals. This can result in reproductive disorders in farm animals as well as in humans. Consequently, detoxification strategies for contaminated crops are crucial for food safety. In this study we have developed a bacterial based detoxification system using a non-pathogen Rhodococcus pyridinivorans K408 strain. Following 5 days treatment of ZEA with R. pyridinivorans K408 strain HPLC analyses showed an 87.21% ZEA-degradation efficiency of the bacterial enzyme systems. In another approach, the strain biotransformation ability has also been confirmed by a bioluminescent version of the yeast estrogen screening system (BLYES), which detected an 81.75% of biodegradability of ZEA, in a good agreement with the chemical analyses. Furthermore, the capacity of R. pyridinivorans to eliminate the estrogenic effects of ZEA was tested by using an immature uterotrophic assay. Prepubertal female rats were treated with vehicle (olive oil), 17ß-estradiol, ZEA (0.1-1-5-10 mg/kg body weight) and LB broth containing 500 mg/l ZEA that has already been incubated with or without Rhodococcus pyridinivorans K408 strain. Uterine weights were measured and the mRNA level changes relating to apelin, aquaporin 5, complement component 2, and calbindin-3 genes were measured by qRT-PCR. These genes represent the major pathways that are affected by estromimetic compounds. Zearalenone feeding significantly increased the uterus weight in a dose dependent manner and at the same time upregulated complement component 2 and calbindin-3 expression as well as decreased apelin and aquaporin 5 mRNA levels comparable to that seen in 17ß-estradiol exposed rats. In contrast, LB broth in which ZEA was incubated with Rhodococcus pyridinivorans K408 prior to the feeding did not display any estrogenic effect neither on uterine weight nor on the expression of estrogen-regulated genes. Consequently, the identification of Rhodococcus pyridinivorans K408 strain in ZEA biodegradation proved to be a very efficient biological tool that is able to eliminate the complete estrogenic effects of ZEA. It is also remarkable that this biotransformation pathway of ZEA did not result in any residual estrogenic effects.