RESUMEN
The assembly of tissue-damaging membrane attack complexes (MACs; C5b-9) is a major mechanism by which excessive complement activation causes diseases. We previously developed a mouse anti-human C6 monoclonal antibody (mAb) 1C9 that selectively inhibits the assembly of MACs in human and non-human primates. In this project, we found that 1C9 also cross-reacted with rat and guinea pig C6, and determined its binding domains on C6 using different truncated C6 proteins. We then humanized the anti-C6 mAb by molecular modeling and complementarity-determining region grafting. After screening a library of 276 humanized variants with different combinations of humanized light and heavy chains in biophysical assays, we identified clone 3713 with the best developability profile, and an increased affinity against C6 when compared with the parental 1C9 mAb. This humanized 3713 mAb inhibited human, monkey, and rat complement-mediated hemolysis in vitro, and more importantly, it significantly reduced complement-mediated hemolysis in vivo in rats. These results demonstrated the successful humanization of the anti-C6 mAb and suggested that the humanized 3713 mAb could be further developed as a new therapeutic that selectively targets MAC for certain complement-mediated pathological conditions.
Asunto(s)
Anticuerpos Monoclonales , Complemento C6 , Hemólisis , Animales , Humanos , Ratas , Cobayas , Ratones , Hemólisis/efectos de los fármacos , Hemólisis/inmunología , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacología , Complemento C6/inmunología , Anticuerpos Monoclonales Humanizados/inmunología , Anticuerpos Monoclonales Humanizados/farmacología , Activación de Complemento/inmunología , Activación de Complemento/efectos de los fármacos , Complejo de Ataque a Membrana del Sistema Complemento/inmunología , Reacciones Cruzadas/inmunologíaRESUMEN
The cytolytic activity of the membrane attack complex (MAC) is pivotal in the complement-mediated elimination of pathogens. Terminal complement pathway (TCP) genes encode the proteins that form the MAC. Although the TCP genes are well conserved within most vertebrate species, the early evolution of the TCP genes is poorly understood. Based on the comparative genomic analysis of the early evolutionary history of the TCP homologs, we evaluated four possible scenarios that could have given rise to the vertebrate TCP. Currently available genomic data support a scheme of complex sequential protein domain gains that may be responsible for the birth of the vertebrate C6 gene. The subsequent duplication and divergence of this vertebrate C6 gene formed the C7, C8α, C8ß, and C9 genes. Compared to the widespread conservation of TCP components within vertebrates, we discovered that C9 has disintegrated in the genomes of galliform birds. Publicly available genome and transcriptome sequencing datasets of chicken from Illumina short read, PacBio long read, and Optical mapping technologies support the validity of the genome assembly at the C9 locus. In this study, we have generated a > 120X coverage whole-genome Chromium 10x linked-read sequencing dataset for the chicken and used it to verify the loss of the C9 gene in the chicken. We find multiple CR1 (chicken repeat 1) element insertions within and near the remnant exons of C9 in several galliform bird genomes. The reconstructed chronology of events shows that the CR1 insertions occurred after C9 gene loss in an early galliform ancestor. Loss of C9 in galliform birds, in contrast to conservation in other vertebrates, may have implications for host-pathogen interactions. Our study of C6 gene birth in an early vertebrate ancestor and C9 gene death in galliform birds provides insights into the evolution of the TCP.
Asunto(s)
Complemento C8 , Complemento C9 , Animales , Pollos/genética , Complemento C6 , Complemento C7/genética , Complemento C8/metabolismo , Complejo de Ataque a Membrana del Sistema Complemento/genética , Proteínas del Sistema Complemento/genética , GenomaRESUMEN
Complement genes encompass a wide array of variants, giving rise to numerous protein isoforms that have often been shown to exhibit clinical significance. Given that these variants have been discovered over a span of 50 y, one challenging consequence is the inconsistency in the terminology used to classify them. This issue is prominently evident in the nomenclature used for complement C6 and C7 variants, for which we observed a great discrepancy between previously published works and variants described in current genome browsers. This report discusses the causes for the discrepancies in C6 and C7 nomenclature and seeks to establish a classification system that would unify existing and future variants. The inconsistency in the methods used to annotate amino acids and the modifications pinpointed in the C6 and C7 primers are some of the factors that contribute greatly to the discrepancy in the nomenclature. Several variants that were classified incorrectly are highlighted in this report, and we showcase first-hand how a unified classification system is important to match previous with current genetic information. Ultimately, we hope that the proposed classification system of nomenclature becomes an incentive for studies on complement variants and their physiological and/or pathological effects.
Asunto(s)
Complemento C6 , Complemento C7 , Complemento C5 , Complemento C6/genética , Complemento C7/genética , Proteínas del Sistema ComplementoRESUMEN
OBJECTIVES: Complement component 6 (C6) deficiency is a very rare genetic defect that leads to significantly diminished synthesis, secretion, or function of C6. In the current report, we demonstrate a previously undescribed, homozygous missense mutation in exon 17 of the C6 gene (c.2545A>G p.Arg849Gly) in a 35-year-old Japanese woman with moyamoya disease and extremely low levels of CH50 (<7.0 U/mL). MATERIALS AND METHODS: The complement gene analysis using hybridization capture-based next generation sequencing was performed. CH50 was determined in patient's plasma mixed with plasma from a healthy donor or purified human C6 protein. Western blot was performed on patient's plasma using polyclonal antibodies against C6, with healthy donor's plasma and purified human C6 protein as positive controls while C6-depleted human serum as a negative control. The carriage of ring finger protein 213 variant (c.14576G>A p.Arg4859Lys), a susceptibility gene for moyamoya disease, was examined by direct sequencing. RESULTS: CH50 mixing test clearly showed a deficiency pattern, being rescued by addition of only 1% healthy donor's plasma or 1 µg/mL purified human C6 protein (1/50-1/100 of physiological concentration). Western blot revealed the absence of C6 protein in the patient's plasma, confirming a quantitative deficiency of C6. The ring finger protein 213 variant was not detected. CONCLUSIONS: Our data implies that unrecognized complement deficiencies would be harbored in cerebrovascular diseases with unknown etiologies.
Asunto(s)
Complemento C6 , Enfermedad de Moyamoya , Adulto , Complemento C6/deficiencia , Complemento C6/genética , Femenino , Enfermedades por Deficiencia de Complemento Hereditario , Humanos , Enfermedad de Moyamoya/complicaciones , Enfermedad de Moyamoya/genética , LinajeRESUMEN
METHODS: Two datasets were used as training and validation cohorts to establish the predictive model. We used three types of screening criteria: background analysis, pathway analysis, and functional analysis provided by the cBioportal website. Fisher's exact test and multivariable logistic regression were performed to screen out related genes. Furthermore, we performed receiver operating characteristic (ROC) and Kaplan-Meier curve analyses to evaluate the correlation between the selected genes and overall survival. RESULT: We screened five genes (KNL1, NRXN1, C6, CCDC169-SOHLH2, and TTN) that were highly related to recurrence of GC. The area under the receiver operating characteristic (ROC) curve was 0.813, which was much higher than that of the baseline model (AUC = 0.699). This result suggested that the mutation of five selected genes had a significant effect on the prediction of recurrence compared with other factors (age, stages, history, etc.). Furthermore, the Kaplan-Meier estimator also revealed that the mutation of five genes positively correlated with patient survival. CONCLUSIONS: The patients who have mutations in these five genes may experience longer survival than those who do not have mutations. This five-gene panel will likely be a practical tool for prognostic evaluation and will provide another possible way for clinicians to determine therapy.
Asunto(s)
Biomarcadores de Tumor/genética , Neoplasias Gástricas/genética , Adulto , Anciano , Algoritmos , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Proteínas de Unión al Calcio/genética , Complemento C6/genética , Conectina/genética , Conjuntos de Datos como Asunto , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Proteínas Asociadas a Microtúbulos/genética , Persona de Mediana Edad , Clasificación del Tumor , Estadificación de Neoplasias , Moléculas de Adhesión de Célula Nerviosa/genética , Pronóstico , Neoplasias Gástricas/patología , Análisis de SupervivenciaRESUMEN
Diagnosis of primary complement deficiencies requires a high index of suspicion. Thus, susceptible patients are often underdiagnosed and untreated. Here, we present a multicenter experience with two novel inborn errors of the classical complement system. This is a retrospective multicenter analysis of computerized medical records of children (<18 years) admitted in the period between 2012 and 2018 at Shaare Zedek Medical Center in Jerusalem and Edmond and Lily Safra Children's Hospital, Tel-Hashomer Medical Center, in Ramat Gan, Israel. Patients were genetically diagnosed by a complementary immune workup. We identified 5 patients (3 males) from four different families harboring two novel mutations in the complement components C6-C8. Genetic mutations were identified by whole-exome sequencing or by sequencing of the coding exons of a single gene based on the findings in the immune workup. Clinical manifestations consisted of meningitis with or without meningococcemia. The immune workup demonstrated nearly absent levels of CH50, compatible with a complement pathway defect. Diagnosis delay ranged between 0 and 30 years. CONCLUSION: Awareness of risk factors for primary complement deficiencies, even at the first infectious episode, should facilitate prompt immune and genetic workup, commencing diagnosis and proper treatment for the patient and family. WHAT IS KNOWN: ⢠Deficiencies in the classical terminal complement components increase susceptibility to invasive meningococcal infections. ⢠Recurrent meningococcal infections mandate a diagnostic workup of the complement system. WHAT IS NEW: ⢠Genetic workup can be utilized for prompt diagnosis of complement deficiencies. ⢠High rates of consanguinity, even in the presence of a single meningococcal infection, should promote immune and genetic workups.
Asunto(s)
Meningitis Meningocócica , Infecciones Meningocócicas , Neisseria meningitidis , Niño , Complemento C6 , Complemento C8/genética , Proteínas del Sistema Complemento/genética , Femenino , Enfermedades por Deficiencia de Complemento Hereditario , Humanos , Masculino , Estudios RetrospectivosRESUMEN
The human complement system provides a first line of defence against pathogens. It requires a well-orchestrated sequential assembly of an array of terminal complement components (C5, C6, C7, C8, and C9), ultimately forming the membrane attack complex (MAC). Although much information about MAC assembly is available, the structure of the soluble C7 has remained elusive. The complement proteins C7 and C6 share very high sequence homology and exhibit several conserved domains, disulphide bridges, and C-mannosylation sites. Here, we used an integrative structural MS-based approach combining native MS, glycopeptide-centric MS, in-gel cross-linking MS (IGX-MS) and structural modelling to describe structural features, including glycosylation, of human serum soluble C7. We compare this data with structural and glycosylation data for human serum C6. The new structural model for C7 shows that it adopts a compact conformation in solution. Although C6 and C7 share many similarities, our data reveals distinct O-, and N-linked glycosylation patterns in terms of location and glycan composition. Cumulatively, our data provide valuable new insight into the structure and proteoforms of C7, solving an essential piece of the puzzle in our understanding of MAC assembly.
Asunto(s)
Complejo de Ataque a Membrana del Sistema Complemento , Proteínas del Sistema Complemento , Complemento C6 , Complemento C7 , Complejo de Ataque a Membrana del Sistema Complemento/metabolismo , Glicosilación , Humanos , Espectrometría de Masas en TándemRESUMEN
The complement component 6 (C6) gene is a component of the membrane attack complex (MAC), which causes rapid lytic destruction of bacteria. MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene stability, including that of immune genes. However, current research on the function of C6 and its regulation by miRNAs is lacking. In the present study, we identified and characterized C6 and a novel miRNA, miR-727 (designated CsC6 and Cse-miR-727, respectively), of the half-smooth tongue sole (Cynoglossus semilaevis) that responded to infection with Vibrio anguillarum, a Gram-negative pathogen of marine fish. The full-length cDNA of CsC6 contained a 256 bp 5' untranslated region (5'-UTR), a 2820 bp open reading frame (ORF) encoding 939 amino acids, and a 205 bp 3'-UTR. SMART analysis showed that CsC6 contains typical C6 domains, including three TSP1 domains, one LDLa domain, one MACPF domain, two CCP domains and two FIMAC domains. CsC6 and Cse-miR-727 are widely expressed in the 13 tissues of half-smooth tongue sole, and their expression in immune tissues is significantly changed after V. anguillarum infection, generally showing an inverse trend. We confirmed that CsC6 was the target gene of Cse-miR-727 using the dual luciferase reporter assay and that Cse-miR-727 regulated CsC6 at the protein level using quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting. The hepatic expression levels of not only the MAC components C7, C8α, C8ß, C8γ and C9 but also the MAPKs, NF-κß, AP-1, IL1ß, IL6 and TNFα, which are involved in many signaling pathways, changed significantly in half-smooth tongue sole following stimulation with the Cse-miR-727 agomir and inhibitor. This evidence suggested that CsC6 could be mediated by Cse-miR-727 to affect MAC assembly and immune signaling pathways in half-smooth tongue soles. To our best knowledge, this study is the first to investigate the regulatory mechanism and immune response of complement genes mediated by miRNAs in fish.
Asunto(s)
Complemento C6/inmunología , Enfermedades de los Peces/inmunología , Proteínas de Peces/inmunología , Peces Planos/inmunología , Hígado/fisiología , MicroARNs/inmunología , Vibriosis/inmunología , Vibrio/fisiología , Animales , Bacteriólisis/genética , Clonación Molecular , Complemento C6/genética , Proteínas de Peces/genética , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Inmunidad Innata , MicroARNs/genéticaRESUMEN
Cross-linking mass spectrometry has developed into an important method to study protein structures and interactions. The in-solution cross-linking workflows involve time and sample consuming steps and do not provide sensible solutions for differentiating cross-links obtained from co-occurring protein oligomers, complexes, or conformers. Here we developed a cross-linking workflow combining blue native PAGE with in-gel cross-linking mass spectrometry (IGX-MS). This workflow circumvents steps, such as buffer exchange and cross-linker concentration optimization. Additionally, IGX-MS enables the parallel analysis of co-occurring protein complexes using only small amounts of sample. Another benefit of IGX-MS, demonstrated by experiments on GroEL and purified bovine heart mitochondria, is the substantial reduction of undesired over-length cross-links compared to in-solution cross-linking. We next used IGX-MS to investigate the complement components C5, C6, and their hetero-dimeric C5b6 complex. The obtained cross-links were used to generate a refined structural model of the complement component C6, resembling C6 in its inactivated state. This finding shows that IGX-MS can provide new insights into the initial stages of the terminal complement pathway.
Asunto(s)
Complemento C5/metabolismo , Complemento C6/metabolismo , Proteínas del Sistema Complemento/metabolismo , Reactivos de Enlaces Cruzados/química , Espectrometría de Masas/métodos , Mitocondrias Cardíacas/metabolismo , Animales , Bovinos , Complemento C5/química , Complemento C6/química , Proteínas del Sistema Complemento/químicaRESUMEN
Neonatal herpes simplex virus (HSV) infection is a devastating disease with high mortality, particularly when disseminated. Studies in adults and children suggest that susceptibility to herpes simplex encephalitis (HSE) may represent phenotypes for inborn errors in toll-like receptor 3 (TLR3) signaling. However, the genetic basis of susceptibility to neonatal HSV including disseminated disease remains unknown. To test the hypothesis that variants in known HSE-susceptible genes as well as genes mediating HSV immunity will be identified in neonatal HSV, we performed an unbiased exome sequencing study in 10 newborns with disseminated, HSE, and skin, eyes, and mouth disease. Determination of potential impact on function was determined by following American College of Medical Genetics and Genomics guidelines. We identified deleterious and potentially deleterious, rare variants in known HSE-related genes including a stop IRF3 variant (disseminated), nonsynonymous variants in TLR3 and TRAF3 (HSE), STAT1 (skin, eyes, and mouth), and DBR1 (disseminated) in our cohort. Novel and rare variants in other immunodeficiency genes or HSV-related immune genes GRB2, RAG2, PRF1, C6, C7, and MSR1 were found in 4 infants. The variant in GRB2, essential for T-lymphocyte cell responses to HSV, is a novel stop variant not found in public databases. In this pilot study, we identified deleterious or potentially deleterious variants in TLR3 pathway and genes that regulate anti-HSV immunity in neonates with HSV including disseminated disease. Larger, definitive studies incorporating functional analysis of genetic variants are required to validate these data and determine the role of immune genetic variants in neonatal HSV susceptibility.
Asunto(s)
Variación Genética , Herpes Simple/genética , Complicaciones Infecciosas del Embarazo/genética , Complemento C6/genética , Complemento C7/genética , Proteínas de Unión al ADN/genética , Femenino , Proteína Adaptadora GRB2/genética , Humanos , Recién Nacido , Factor 3 Regulador del Interferón/genética , Masculino , Proteínas Nucleares/genética , Perforina/genética , Proyectos Piloto , Embarazo , ARN Nucleotidiltransferasas/genética , Factor de Transcripción STAT1/genética , Receptores Depuradores de Clase A/genética , Factor 3 Asociado a Receptor de TNF/genética , Receptor Toll-Like 3/genética , Secuenciación del ExomaRESUMEN
Historically, the membrane attack complex, composed of complement components C5b-9, has been connected to lytic cell death and implicated in secondary injury after a CNS insult. However, studies to date have utilized either non-littermate control rat models, or mouse models that lack significant C5b-9 activity. To investigate what role C5b-9 plays in spinal cord injury and recovery, we generated littermate PVG C6 wildtype and deficient rats and tested functional and histological recovery after moderate contusion injury using the Infinite Horizon Impactor. We compare the effect of C6 deficiency on recovery of locomotor function and histological injury parameters in PVG rats under two conditions: (1) animals maintained as separate C6 WT and C6-D homozygous colonies; and (2) establishment of a heterozygous colony to generate C6 WT and C6-D littermate controls. The results suggest that maintenance of separate homozygous colonies is inadequate for testing the effect of C6 deficiency on locomotor and histological recovery after SCI, and highlight the importance of using littermate controls in studies involving genetic manipulation of the complement cascade.
Asunto(s)
Complemento C6/deficiencia , Enfermedades por Deficiencia de Complemento Hereditario/complicaciones , Traumatismos de la Médula Espinal/metabolismo , Traumatismos de la Médula Espinal/fisiopatología , Animales , Conducta Animal , Complemento C6/genética , Complejo de Ataque a Membrana del Sistema Complemento/genética , Complejo de Ataque a Membrana del Sistema Complemento/metabolismo , Modelos Animales de Enfermedad , Femenino , Genotipo , Proteína Ácida Fibrilar de la Glía/metabolismo , Sustancia Gris/citología , Sustancia Gris/metabolismo , Enfermedades por Deficiencia de Complemento Hereditario/genética , Heterocigoto , Locomoción , Masculino , Proteína Básica de Mielina/metabolismo , Ratas Mutantes , Selección Artificial , Traumatismos de la Médula Espinal/etiología , Traumatismos de la Médula Espinal/genética , Vértebras Torácicas/lesiones , Sustancia Blanca/citología , Sustancia Blanca/metabolismoRESUMEN
Background: Terminal complement component deficiencies are risk factors for neisserial infections. Objective: To review the clinical characteristics, the diagnosis and the management of patients with a terminal complement component deficiency. Methods: Pertinent articles were selected and reviewed in relation to a case presentation of C6 deficiency. Results: A case of a 56-year old patient with a history of meningitis, chronic rash, and C6 deficiency was presented, followed by discussion of clinical characteristics, diagnosis, and management of terminal complement component deficiencies. Clinical pearls and pitfalls were reviewed for the practicing allergist/immunologist and fellow-in-training. Conclusion: C6 deficiency is the most common terminal complement component deficiency and can present later in age with N. meningitidis infections. Patients can be screened for terminal complement component deficiency by checking CH50.
Asunto(s)
Envejecimiento/fisiología , Complemento C6/deficiencia , Complemento C6/genética , Enfermedades por Deficiencia de Complemento Hereditario/diagnóstico , Meningitis Meningocócica/diagnóstico , Vacunas Meningococicas/inmunología , Neisseria meningitidis/fisiología , Profilaxis Antibiótica , Ensayo de Actividad Hemolítica de Complemento , Femenino , Fibronectinas/análisis , Enfermedades por Deficiencia de Complemento Hereditario/complicaciones , Humanos , Meningitis Meningocócica/etiología , Meningitis Meningocócica/prevención & control , Persona de Mediana Edad , Proteínas Recombinantes/análisisRESUMEN
RATIONALE: Late complement deficiency increases susceptibility to meningococcal disease and recurrent infections. In Korea, 5 case reports have described meningococcal disease with complement deficiency. However, C6 deficiency has not been described previously. PATIENT CONCERNS: A 21-year-old police trainee presented with recurrent meningococcal meningitis. He was housed in communal living quarters until 20 days before the initial symptom onset. DIAGNOSIS: He was diagnosed with meningococcal meningitis with C6 deficiency. INTERVENTIONS: He was treated with intravenous ceftriaxone. An additional dose of quadrivalent meningococcal conjugate vaccine was administered after discharge. OUTCOMES: He was discharged without complications. LESSONS: Screening for complement deficiency is necessary in patients with a history of recurrent meningococcal infections to provide appropriate care and prevent recurrent infections.
Asunto(s)
Complemento C6/deficiencia , Meningitis Meningocócica/diagnóstico , Complemento C6/inmunología , Exantema/etiología , Fiebre/etiología , Cefalea/etiología , Humanos , Masculino , Meningitis Meningocócica/tratamiento farmacológico , Meningitis Meningocócica/inmunología , Vacunas Meningococicas/normas , Vacunas Meningococicas/uso terapéutico , Recurrencia , República de Corea , Adulto JovenRESUMEN
Over the first days of polymicrobial sepsis, there is robust activation of the innate immune system, causing the appearance of proinflammatory cytokines and chemokines, along with the appearance of extracellular histones, which are highly proinflammatory and prothrombotic. In the current study, we studied different innate immune responses in mice with knockout (KO) of complement protein 6 (C6). Polymorphonuclear neutrophils (PMNs) from these KO mice had defective innate immune responses, including defective expression of surface adhesion molecules, generation of superoxide anion, and appearance of reactive oxygen species and histone release after activation of PMNs, along with defective phagocytosis. In addition, in C6-/- mice, the NLRP3 inflammasome was defective both in PMNs and in macrophages. When these KO mice were subjected to polymicrobial sepsis, their survival was improved, associated with reduced levels in the plasma of proinflammatory cytokines and chemokines and lower levels of histones in plasma. In addition, sepsis-induced cardiac dysfunction was attenuated in these KO mice. In a model of acute lung injury induced by LPS, C6-/- mice showed reduced PMN buildup and less lung epithelial/endothelial cell dysfunction (edema and hemorrhage). These data indicate that C6-/- mice have reduced innate immune responses that result in less organ injury and improved survival after polymicrobial sepsis.
Asunto(s)
Lesión Pulmonar Aguda/inmunología , Cardiomiopatías/inmunología , Coinfección/inmunología , Complemento C6/metabolismo , Inmunidad Innata , Sepsis/inmunología , Lesión Pulmonar Aguda/diagnóstico , Lesión Pulmonar Aguda/patología , Animales , Cardiomiopatías/diagnóstico , Cardiomiopatías/patología , Coinfección/complicaciones , Coinfección/diagnóstico , Coinfección/patología , Complemento C6/genética , Modelos Animales de Enfermedad , Humanos , Inflamasomas/inmunología , Inflamasomas/metabolismo , Masculino , Ratones , Ratones Noqueados , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Neutrófilos/inmunología , Neutrófilos/metabolismo , Sepsis/complicaciones , Sepsis/diagnóstico , Sepsis/genética , Índice de Severidad de la EnfermedadRESUMEN
Membrane attack complexes (MACs; C5b-9) assembled after complement activation can directly injure self-tissues, leading to various diseases. Eculizumab, a monoclonal antibody (mAb) against complement component C5, is being used in the clinic to treat diseases in which MAC-mediated tissue damage is a primary cause. However, C5 is not a selective target for MAC assembly inhibition, and some patients respond incompletely or not at all to the eculizumab treatment. Therefore, C6, the next essential component in the terminal pathway of complement activation, may be an alternative target for the selective inhibition of MAC formation. Surprisingly, few reports describe a functional blockade of C6 using a specific mAb. Here, we report the development of an anti-human C6 mAb (clone 1C9) that recognizes C6 both in free circulation and within C5b6 complexes. This mAb blocked C7 binding to C5b6 complexes and consequently inhibited MAC formation and protected affected paroxysmal nocturnal hemoglobinuria patient red blood cells from MAC-mediated damage in vitro. In addition, this mAb cross-reacts with rhesus monkey but not mouse complement C6. Finally, 1C9 significantly reduced human complement-mediated intravascular hemolysis in vivo in a mouse model. These results suggest that the anti-C6 mAb holds promise as a new therapeutic agent that selectively targets MAC for many complement-mediated pathological conditions.
Asunto(s)
Complemento C6 , Complejo de Ataque a Membrana del Sistema Complemento , Animales , Activación de Complemento , Complemento C5 , Hemólisis , Humanos , RatonesRESUMEN
BACKGROUND: Endometriosis is a disease that shows auto-immune and chronic characteristics, suggesting a role for proteins mediating immune interactions in its pathophysiology. The aim was to evaluate C3a and C5a for their role in inflammatory responses and C6 as the down-stream interactor following our previous findings on C5 mRNA expression changes in endometriosis [1]. METHODS: Sera from 71 endometriosis patients and 77 women without endometriosis were taken. While the samples were taken only once from the controls, the patient samples were taken before, in 1st and in 7th days after laparoscopy. Levels of complement proteins C3a, C5a and C6 were measured with ELISA assays. MPV (Mean Platelet Volume), CRP (C-Reactive Protein) and NLR (Neutrophil-to-Leukocyte Ratio) were also analyzed from the retrospective data. RESULTS: C6 levels of early-stage patients at postoperative 1st day were significantly higher than controls. Patients with high MPV measurements had significantly higher C3a (p < 0.0001) and C6 (p < 0.05) levels than controls at all times of measurement. CONCLUSIONS: C6, an integral component of the membrane attack complex (MAC), could play a role at early disease-stage. The changes in levels of complement proteins and their relation to high MPV levels suggest a broader area of interplay for immune interactors in endometriosis. Although a bigger and longitudinal study design is needed to obtain more accurate results to evaluate these proteins as potential biomarkers, an important role of complement system within the pathophysiology of endometriosis is apparent.
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Biomarcadores/sangre , Plaquetas/patología , Complemento C3a/metabolismo , Complemento C5a/metabolismo , Complemento C6/metabolismo , Endometriosis/patología , Adolescente , Adulto , Endometriosis/sangre , Endometriosis/inmunología , Ensayo de Inmunoadsorción Enzimática/métodos , Femenino , Humanos , Estudios Longitudinales , Persona de Mediana Edad , Estudios Retrospectivos , Adulto JovenRESUMEN
The purpose of this study was to identify a membrane-bound complement inhibitor that could be overexpressed on retinal pigment epithelial cells (RPE) providing a potential therapy for age-related macular degeneration (AMD). This type of therapy may allow replacement of damaged RPE with cells that are able to limit complement activation in the retina. Complement Receptor 1 (CR1) is a membrane-bound complement inhibitor commonly found on erythrocytes and immune cells. In this study, QPCR and flow cytometry data demonstrated that CR1 is not well-expressed by RPE, indicating that its overexpression may provide extra protection from complement activation. To screen CR1 for this ability, a stable CR1-expressing ARPE19 line was created using a combination of antibiotic selection and FACS. Cell-based assays were used to demonstrate that addition of CR1 inhibited deposition of complement proteins C3b and C6 on the transfected line. In the end, this study identifies CR1 as a complement inhibitor that may be overexpressed on stem cell-derived RPE to create a potential "enhanced" cell therapy for AMD. A combination cell/complement therapy may create transplantable RPE better suited to avoid complement-mediated lysis and limit chronic inflammation in the retina.
Asunto(s)
Células Epiteliales/inmunología , Degeneración Macular/inmunología , Receptores de Complemento 3b/inmunología , Retina/inmunología , Epitelio Pigmentado de la Retina/inmunología , Pigmentos Retinianos/inmunología , Línea Celular , Activación de Complemento/inmunología , Complemento C3b/inmunología , Complemento C6/inmunología , Eritrocitos/inmunología , HumanosRESUMEN
BACKGROUND: X-linked agammaglobulinemia (XLA) is a primary immunodeficiency disorder caused by germline mutations in the Bruton tyrosine kinase (BTK) gene on X chromosome. These mutations disturb B-cell development, decrease immunoglobulin levels, increase susceptibility to infection or neoplasms, and increase the risk of developing colorectal cancer (CRC). For occasional cases of CRC have been reported in XLA patients, low levels of B lymphocytes and immunoglobulins induced by congenital immune disorder make them more susceptible to drug-related toxicities (DRT). Therefore, gene sequencing, therapeutic drug monitoring and any possible measurement to predict DRT should be considered before determining the course of chemotherapy for XLA patients with CRC. CASE PRESENTATION: In this study, we reported a 21-year-old male who developed metastatic CRC in the context of XLA. Since the whole exome sequencing and therapeutic drug monitoring did not reveal any predictive markers of DRT, we applied standard first-line chemotherapy to the patient. However, progressive disease occurred after the fifth treatment cycle. Therefore, the administration of oxaliplatin was changed to irinotecan as second-line therapy. After that, the patient firstly suffered from severe hypocalcemia and eventually died due to metastatic CRC after the eighth treatment cycle. The overall survival time was 7.5 months. CONCLUSIONS: This study reported the first written record of a Chinese XLA patient with metastatic CRC and severe hypocalcemia. Whole exome sequencing and bioinformatic analysis indicated the somatic mutations in ABCA6, C6 and PAX3 genes might contribute to the early-onset and metastasis CRC. Besides, a number of germline mutations in genes related to calcium metabolism (CACNA2D4, CD36, etc.) and the administration of irinotecan were speculated to be the causes of severe hypocalcemia. We therefore suggested that in order to avoid severe DRT, clinicians should take genetic background and therapeutic drug monitoring into consideration while planning chemotherapy treatment for XLA patients with CRC.
Asunto(s)
Agammaglobulinemia Tirosina Quinasa/genética , Agammaglobulinemia/genética , Neoplasias Colorrectales/genética , Enfermedades Genéticas Ligadas al Cromosoma X/genética , Predisposición Genética a la Enfermedad/genética , Hipocalcemia/complicaciones , Irinotecán/administración & dosificación , Transportadoras de Casetes de Unión a ATP/genética , Adulto , Agammaglobulinemia/diagnóstico por imagen , Pueblo Asiatico , Linfocitos B , Canales de Calcio Tipo L/genética , Capecitabina/administración & dosificación , Capecitabina/uso terapéutico , Neoplasias Colorrectales/tratamiento farmacológico , Complemento C6/genética , Monitoreo de Drogas , Quimioterapia , Estudios de Asociación Genética , Enfermedades Genéticas Ligadas al Cromosoma X/diagnóstico por imagen , Humanos , Hipocalcemia/inducido químicamente , Inmunoglobulinas , Irinotecán/uso terapéutico , Masculino , Mutación , Oxaliplatino/uso terapéutico , Factor de Transcripción PAX3/genética , Secuenciación del Exoma , Adulto JovenRESUMEN
Exosomes are vehicles in the body fluid that participate in many biological processes, especially immune responses. In this study, we employed comparative proteome analysis to investigate the roles of serum exosomes during viral infection in neonates using porcine epidemic diarrhea virus (PEDV), a devastating enteric virus in newborn piglets, as a model virus. Serum exosomes were first isolated from newborn piglets infected with PEDV or mock-infected newborn piglets, followed by label-free LC-MS/MS-based comparative quantitative proteomic analysis. Among the 441 detected proteins, 10 complement proteins were found in the serum exosomes, and significantly decreased expression levels of the C3, C6, and CFB complements were measured in PEDV-infected serum exosomes compared to those in mock-infected serum exosomes. After confirmation by Western blot, we then investigated the function of these exosomes in PEDV infection and discovered that exosomes from mock-infected newborn piglets restricted PEDV infection. However, this inhibition disappeared after the exosomes were heat-inactivated, suggesting that complements are key antiviral molecules. Our findings improve the understanding of antiviral responses mediated by exosomes in neonatal piglets and facilitate the discovery of novel antiviral drugs.
Asunto(s)
Complemento C3/genética , Complemento C6/genética , Factor B del Complemento/genética , Infecciones por Coronavirus/inmunología , Exosomas/inmunología , Enfermedades de los Porcinos/inmunología , Animales , Animales Recién Nacidos , Cromatografía Liquida , Complemento C3/metabolismo , Complemento C6/metabolismo , Factor B del Complemento/metabolismo , Infecciones por Coronavirus/genética , Infecciones por Coronavirus/patología , Infecciones por Coronavirus/virología , Exosomas/química , Cadenas Pesadas de Inmunoglobulina/sangre , Cadenas Pesadas de Inmunoglobulina/genética , Redes y Vías Metabólicas/genética , Redes y Vías Metabólicas/inmunología , Virus de la Diarrea Epidémica Porcina/inmunología , Virus de la Diarrea Epidémica Porcina/patogenicidad , Proteómica/métodos , Porcinos , Enfermedades de los Porcinos/genética , Enfermedades de los Porcinos/patología , Enfermedades de los Porcinos/virología , Espectrometría de Masas en TándemRESUMEN
Atypical hemolytic uremic syndrome (aHUS) is a form of thrombotic microangiopathy (TMA) caused by dysregulated complement activation. Clinically, aHUS is effectively treated by an anti-C5 monoclonal antibody (mAb) but whether the disease is mediated by the C5a receptor (C5aR) or C5b-9 pathway, or both, is unknown. Here we address this in a factor H mutant mouse (FHR/R) which developed complement-mediated TMA as well as macrovascular thrombosis caused by an aHUS-related factor H point mutation (mouse W1206R, corresponding to human W1183R). C5 deficiency and anti-C5 mAb treatment blocked all disease manifestations in FHR/R mice. C5aR1 gene deficiency prevented macrovascular thrombosis in various organs but did not improve survival or reduce renal TMA. Conversely, C6 or C9 deficiency significantly improved survival and markedly diminished renal TMA but did not prevent macrovascular thrombosis. Interestingly, as they aged both FHR/R C6-/- and FHR/R C9-/- mice developed glomerular disease reminiscent of C3 glomerulonephritis. Thus, C5aR and C5b-9 pathways drove different aspects of disease in FHR/R mice with the C5aR pathway being responsible for macrovascular thrombosis and chronic inflammatory injury while the C5b-9 pathway caused renal TMA. Our data provide new understanding of the pathogenesis of complement-mediated TMA and macrovascular thrombosis in FHR/R mice and suggest that C5 blockade is more effective for the treatment of aHUS than selectively targeting the C5aR or C5b-9 pathway alone.