RESUMO
Paroxysmal nocturnal hemoglobinuria (PNH) is a rare hemolytic disease driven by impaired complement regulation. Mutations in genes encoding the enzymes that build the GPI anchors are causative, with somatic mutations in the PIG-A gene occurring most frequently. As a result, the important membrane-bound complement regulators CD55 and CD59 are missing on the affected hematopoietic stem cells and their progeny, rendering those cells vulnerable to complement attack. Immune escape mechanisms sparing affected PNH stem cells from removal are suspected in the PNH pathogenesis, but molecular mechanisms have not been elucidated. We hypothesized that exuberant complement activity in PNH results in enhanced immune checkpoint interactions, providing a molecular basis for the potential immune escape in PNH. In a series of PNH patients, we found increased expression levels of the checkpoint ligand programmed death-ligand 1 (PD-L1) on granulocytes and monocytes, as well as in the plasma of PNH patients. Mechanistically, we demonstrate that complement activation leading to the decoration of particles/cells with C3- and/or C4-opsonins increased PD-L1 expression on neutrophils and monocytes as shown for different in vitro models of classical or alternative pathway activation. We further establish in vitro that complement inhibition at the level of C3, but not C5, inhibits the alternative pathway-mediated upregulation of PD-L1 and show by means of soluble PD-L1 that this observation translates into the clinical situation when PNH patients are treated with either C3 or C5 inhibitors. Together, the presented data show that the checkpoint ligand PD-L1 is increased in PNH patients, which correlates with proximal complement activation.
Assuntos
Antígeno B7-H1/metabolismo , Ativação do Complemento/imunologia , Complemento C3/antagonistas & inibidores , Complemento C5/antagonistas & inibidores , Hemoglobinúria Paroxística/patologia , Antígeno B7-H1/sangue , Antígenos CD55/genética , Antígenos CD59/genética , Complemento C3/imunologia , Complemento C5/imunologia , Granulócitos/metabolismo , Células-Tronco Hematopoéticas/citologia , Hemoglobinúria Paroxística/imunologia , Humanos , Evasão da Resposta Imune/imunologia , Proteínas de Membrana/genética , Monócitos/metabolismoRESUMO
BACKGROUND & AIMS: Acute liver failure (ALF) is a life-threatening condition with limited treatment alternatives. ALF pathogenesis seemingly involves the complement system. However, no complement-targeted intervention has been clinically applied. In this study, we aimed to investigate the potential of Complement-5 (C5)-targeted ALF treatment. METHODS: ALF was induced in C5-knockout (KO, B10D2/oSn) mice and their wild-type (WT) counterparts (B10D2/nSn) through intraperitoneal lipopolysaccharide (LPS) and d-galactosamine (D-GalN) administration. Thereafter, monoclonal anti-C5 antibody (Ab) or control immunoglobulin was administered intravenously. Furthermore, a selective C5a-receptor (C5aR) antagonist was administered to WT mice to compare its efficacy with that of anti-C5-Ab-mediated total C5 inhibition. We clarified the therapeutic effect of delayed anti-C5-Ab administration after LPS/D-GalN challenge. We also assessed the efficacy of anti-C5-Ab in another ALF model, using concanavalin-A. RESULTS: Liver injury was evident 6 hours after LPS/D-GalN administration. C5-KO and anti-C5-Ab treatment significantly improved overall animal survival and significantly reduced serum transaminase and high-mobility group box-1 release with decreased histological tissue damage. This improvement was characterized by significantly reduced CD41+ platelet aggregation, maintained F4/80+ cells, and less infiltration of CD11+/Ly6-G+ cells with lower cytokine/chemokine expression. Furthermore, C5-KO and anti-C5-Ab downregulated tumor necrosis factor-α production by macrophages before inducing marked liver injury. Moreover, single-stranded-DNA cells and caspase activation were reduced, indicating significant attenuation of apoptosis. Anti-C5-Ab treatment protected the liver more effectively than the C5aR antagonist, and its delayed doses were hepatoprotective. In addition, anti-C5-Ab treatment was effective against concanavalin-A-induced ALF. CONCLUSIONS: C5 inhibition effectively suppresses progression to ALF in mice models of fulminant hepatitis, serving as a new potential treatment strategy for ALF.
Assuntos
Anticorpos Monoclonais/farmacologia , Complemento C5/antagonistas & inibidores , Modelos Animais de Doenças , Falência Hepática Aguda/prevenção & controle , Macrófagos/efeitos dos fármacos , Necrose Hepática Massiva/complicações , Animais , Apoptose , Complemento C5/imunologia , Progressão da Doença , Falência Hepática Aguda/etiologia , Falência Hepática Aguda/patologia , Macrófagos/imunologia , Masculino , Camundongos , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Growing clinical evidence has implicated complement as a pivotal driver of COVID-19 immunopathology. Deregulated complement activation may fuel cytokine-driven hyper-inflammation, thrombotic microangiopathy and NET-driven immunothrombosis, thereby leading to multi-organ failure. Complement therapeutics have gained traction as candidate drugs for countering the detrimental consequences of SARS-CoV-2 infection. Whether blockade of terminal complement effectors (C5, C5a, or C5aR1) may elicit similar outcomes to upstream intervention at the level of C3 remains debated. Here we compare the efficacy of the C5-targeting monoclonal antibody eculizumab with that of the compstatin-based C3-targeted drug candidate AMY-101 in small independent cohorts of severe COVID-19 patients. Our exploratory study indicates that therapeutic complement inhibition abrogates COVID-19 hyper-inflammation. Both C3 and C5 inhibitors elicit a robust anti-inflammatory response, reflected by a steep decline in C-reactive protein and IL-6 levels, marked lung function improvement, and resolution of SARS-CoV-2-associated acute respiratory distress syndrome (ARDS). C3 inhibition afforded broader therapeutic control in COVID-19 patients by attenuating both C3a and sC5b-9 generation and preventing FB consumption. This broader inhibitory profile was associated with a more robust decline of neutrophil counts, attenuated neutrophil extracellular trap (NET) release, faster serum LDH decline, and more prominent lymphocyte recovery. These early clinical results offer important insights into the differential mechanistic basis and underlying biology of C3 and C5 inhibition in COVID-19 and point to a broader pathogenic involvement of C3-mediated pathways in thromboinflammation. They also support the evaluation of these complement-targeting agents as COVID-19 therapeutics in large prospective trials.
Assuntos
Betacoronavirus/patogenicidade , Complemento C3/antagonistas & inibidores , Complemento C5/antagonistas & inibidores , Inativadores do Complemento/uso terapêutico , Infecções por Coronavirus/tratamento farmacológico , Fatores Imunológicos/uso terapêutico , Pneumonia Viral/tratamento farmacológico , Síndrome do Desconforto Respiratório/tratamento farmacológico , Anticorpos Monoclonais Humanizados/uso terapêutico , Biomarcadores/sangue , Proteína C-Reativa/metabolismo , COVID-19 , Estudos de Coortes , Ativação do Complemento/efeitos dos fármacos , Complemento C3/genética , Complemento C3/imunologia , Complemento C5/genética , Complemento C5/imunologia , Infecções por Coronavirus/complicações , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Armadilhas Extracelulares/efeitos dos fármacos , Feminino , Expressão Gênica , Humanos , Interleucina-6/metabolismo , Masculino , Pessoa de Meia-Idade , Neutrófilos/efeitos dos fármacos , Neutrófilos/imunologia , Neutrófilos/virologia , Pandemias , Peptídeos Cíclicos/uso terapêutico , Pneumonia Viral/complicações , Pneumonia Viral/imunologia , Pneumonia Viral/virologia , Síndrome do Desconforto Respiratório/complicações , Síndrome do Desconforto Respiratório/imunologia , Síndrome do Desconforto Respiratório/virologia , SARS-CoV-2 , Índice de Gravidade de DoençaRESUMO
As a novel alternative to established surface display or combinatorial chemistry approaches for the discovery of therapeutic peptides, we present a method for the isolation of small, cysteine-rich domains from bovine antibody ultralong complementarity-determining regions (CDRs). We show for the first time that isolated bovine antibody knob domains can function as autonomous entities by binding antigen outside the confines of the antibody scaffold. This yields antibody fragments so small as to be considered peptides, each stabilised by an intricate, bespoke arrangement of disulphide bonds. For drug discovery, cow immunisations harness the immune system to generate knob domains with affinities in the picomolar to low nanomolar range, orders of magnitude higher than unoptimized peptides from naïve library screening. Using this approach, knob domain peptides that tightly bound Complement component C5 were obtained, at scale, using conventional antibody discovery and peptide purification techniques.
Assuntos
Anticorpos/química , Dissulfetos/isolamento & purificação , Domínios de Imunoglobulina , Fragmentos de Peptídeos/isolamento & purificação , Domínios e Motivos de Interação entre Proteínas , Animais , Anticorpos/imunologia , Anticorpos/metabolismo , Afinidade de Anticorpos , Formação de Anticorpos , Especificidade de Anticorpos , Antígenos/genética , Antígenos/imunologia , Linfócitos B/fisiologia , Bovinos , Complemento C5/química , Complemento C5/genética , Complemento C5/imunologia , Regiões Determinantes de Complementaridade/química , Regiões Determinantes de Complementaridade/genética , Regiões Determinantes de Complementaridade/imunologia , Dissulfetos/química , Dissulfetos/imunologia , Mapeamento de Epitopos/métodos , Humanos , Imunização , Domínios de Imunoglobulina/genética , Modelos Moleculares , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/imunologia , Domínios e Motivos de Interação entre Proteínas/genéticaRESUMO
INTRODUCTION: Acetylcholine receptor antibody-positive generalized myasthenia gravis (gMG) is effectively treated with symptomatic and immunosuppressive drugs but a proportion of patients has a persistent disease and severe adverse events (AEs). The unmet medical needs are specific immunosuppression and AE lowering. Eculizumab blocks C5 protecting neuromuscular junction from the destructive autoantibody effects. Phase II (Study C08-001) and III (ECU-MG-301) studies, with the open-label extension (ECU-MG-302), demonstrated eculizumab efficacy and safety in refractory gMG patients. AREAS COVERED: We provide an overview of eculizumab biological features, clinical efficacy, and safety in gMG patients, highlighting our perspective on the drug positioning in the MG treatment algorithm. EXPERT OPINION: Eculizumab has the potential to significantly change the immunosuppressive approach in gMG offering the opportunity to avoid or delay corticosteroids' use due to its speed and selective mechanism of action. Eculizumab prescription will depend on: 1. ability to modify the natural disease course; 2. sustainability in the clinical practice (cost/effectiveness ratio); 3. drug-induced AE reduction. At present we are missing a controlled study on its use as a first-line treatment. We think that immunosuppression in MG will change significantly in the next years by adopting more focused 'Precision Medicine' approaches, and Eculizumab seems to satisfy such a promise.
Assuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Inativadores do Complemento/uso terapêutico , Miastenia Gravis/tratamento farmacológico , Anticorpos Monoclonais Humanizados/imunologia , Anticorpos Monoclonais Humanizados/metabolismo , Ensaios Clínicos como Assunto , Complemento C5/imunologia , Inativadores do Complemento/imunologia , Inativadores do Complemento/metabolismo , Meia-Vida , Humanos , Miastenia Gravis/patologia , Resultado do TratamentoRESUMO
Complement activation as a driver of pathology in myasthenia gravis (MG) has been appreciated for decades. The terminal complement component [membrane attack complex (MAC)] is found at the neuromuscular junctions of patients with MG. Animals with experimental autoimmune MG are dependent predominantly on an active complement system to develop weakness. Mice deficient in intrinsic complement regulatory proteins demonstrate a significant increase in the destruction of the neuromuscular junction. As subtypes of MG have been better defined, it has been appreciated that acetylcholine receptor antibody-positive disease is driven by complement activation. Preclinical assessments have confirmed that complement inhibition would be a viable therapeutic approach. Eculizumab, an antibody directed toward the C5 component of complement, was demonstrated to be effective in a Phase 3 trial with subsequent approval by the Federal Drug Administration of the United States and other worldwide regulatory agencies for its use in acetylcholine receptor antibody-positive MG. Second- and third-generation complement inhibitors are in development and approaching pivotal efficacy evaluations. This review will summarize the history and present the state of knowledge of this new therapeutic modality.
Assuntos
Ativação do Complemento/efeitos dos fármacos , Complemento C5/antagonistas & inibidores , Inativadores do Complemento/uso terapêutico , Miastenia Gravis/tratamento farmacológico , Animais , Anticorpos Monoclonais Humanizados/uso terapêutico , Complemento C5/imunologia , Complemento C5/metabolismo , Inativadores do Complemento/efeitos adversos , Modelos Animais de Doenças , Humanos , Miastenia Gravis/imunologia , Miastenia Gravis/metabolismo , Resultado do TratamentoRESUMO
Maintenance of homeostasis at body barriers that are constantly challenged by microbes, toxins and potentially bioactive (macro)molecules requires complex, highly orchestrated mechanisms of protection. Recent discoveries in respiratory research have shed light on the unprecedented role of airway epithelial cells (AEC), which, besides immune cells homing to the lung, also significantly contribute to host defence by expressing membrane-bound and soluble pattern recognition receptors (sPRR). Recent evidence suggests that distinct, evolutionary ancient, sPRR secreted by AEC might become activated by usually innocuous proteins, commonly referred to as allergens. We here provide a systematic overview on sPRR detectable in the mucus lining of AEC. Some of them become actively produced and secreted by AECs (like the pentraxins C-reactive protein and pentraxin 3; the collectins mannose binding protein and surfactant proteins A and D; H-ficolin; serum amyloid A; and the complement components C3 and C5). Others are elaborated by innate and adaptive immune cells such as monocytes/macrophages and T cells (like the pentraxins C-reactive protein and pentraxin 3; L-ficolin; serum amyloid A; and the complement components C3 and C5). Herein we discuss how sPRRs may contribute to homeostasis but sometimes also to overt disease (e.g. airway hyperreactivity and asthma) at the alveolar-air interface.
Assuntos
Asma/imunologia , Hiper-Reatividade Brônquica/imunologia , Proteína C-Reativa/imunologia , Homeostase/imunologia , Receptores de Reconhecimento de Padrão/imunologia , Mucosa Respiratória/imunologia , Alérgenos/administração & dosagem , Animais , Asma/genética , Asma/patologia , Hiper-Reatividade Brônquica/genética , Hiper-Reatividade Brônquica/patologia , Proteína C-Reativa/genética , Colectinas/genética , Colectinas/imunologia , Complemento C3/genética , Complemento C3/imunologia , Complemento C5/genética , Complemento C5/imunologia , Células Epiteliais/imunologia , Células Epiteliais/patologia , Regulação da Expressão Gênica , Homeostase/genética , Humanos , Lectinas/genética , Lectinas/imunologia , Receptores de Reconhecimento de Padrão/genética , Mucosa Respiratória/patologia , Proteína Amiloide A Sérica/genética , Proteína Amiloide A Sérica/imunologia , Componente Amiloide P Sérico/genética , Componente Amiloide P Sérico/imunologiaRESUMO
Better understanding of roles of complement in pathology has fuelled an explosion of interest in complement-targeted therapeutics. The C5-blocking monoclonal antibody (mAb) eculizumab, the first of the new wave of complement blocking drugs, was FDA approved for treatment of Paroxysmal Nocturnal Hemoglobinuria in 2007; its expansion into other diseases has been slow and remains restricted to rare and ultra-rare diseases such as atypical hemolytic uremic syndrome. The success of eculizumab has provoked other Pharma to follow this well-trodden track and made C5 blockade the busiest area of complement drug development. C5 blockade inhibits generation of C5a and C5b, the former an anaphylatoxin, the latter the nidus for formation of the pro-inflammatory membrane attack complex. In order to use anti-complement drugs in common complement-driven diseases, more affordable and equally effective therapeutics are needed. To address this, we explored complement inhibition downstream of C5. Novel blocking mAbs targeting C7 and/or the C5b-7 complex were generated, identified using high throughput functional assays and specificity confirmed by immunochemical assays and surface plasmon resonance (SPR). Selected mAbs were tested in rodents to characterize pharmacokinetics, and therapeutic capacity. Administration of a mouse C7-selective mAb to wildtype mice, or a human C7 specific mAb to C7-deficient mice reconstituted with human C7, completely inhibited serum lytic activity for >48 h. The C5b-7 complex selective mAb 2H2, most active in rat serum, efficiently inhibited serum lytic activity in vivo for over a week from a single low dose (10 mg/kg); this mAb effectively blocked disease and protected muscle endplates from destruction in a rat myasthenia model. Targeting C7 and C7-containing terminal pathway intermediates is an innovative therapeutic approach, allowing lower drug dose and lower product cost, that will facilitate the expansion of complement therapeutics to common diseases.
Assuntos
Anticorpos Monoclonais/imunologia , Complemento C5/imunologia , Animais , Anticorpos Monoclonais Humanizados/imunologia , Antineoplásicos Imunológicos/imunologia , Síndrome Hemolítico-Urêmica Atípica/imunologia , Ativação do Complemento/imunologia , Complexo de Ataque à Membrana do Sistema Complemento/imunologia , Hemoglobinúria Paroxística/imunologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , RatosRESUMO
Rationale: Pulmonary hypertension (PH) is a life-threatening cardiopulmonary disorder in which inflammation and immunity have emerged as critical early pathogenic elements. Although proinflammatory processes in PH and pulmonary arterial hypertension (PAH) are the focus of extensive investigation, the initiating mechanisms remain elusive.Objectives: We tested whether activation of the complement cascade is critical in regulating proinflammatory and pro-proliferative processes in the initiation of experimental hypoxic PH and can serve as a prognostic biomarker of outcome in human PAH.Methods: We used immunostaining of lung tissues from experimental PH models and patients with PAH, analyses of genetic murine models lacking specific complement components or circulating immunoglobulins, cultured human pulmonary adventitial fibroblasts, and network medicine analysis of a biomarker risk panel from plasma of patients with PAH.Measurements and Main Results: Pulmonary perivascular-specific activation of the complement cascade was identified as a consistent critical determinant of PH and PAH in experimental animal models and humans. In experimental hypoxic PH, proinflammatory and pro-proliferative responses were dependent on complement (alternative pathway and component 5), and immunoglobulins, particularly IgG, were critical for activation of the complement cascade. We identified Csf2/GM-CSF as a primary complement-dependent inflammatory mediator. Furthermore, using network medicine analysis of a biomarker risk panel from plasma of patients with PAH, we demonstrated that complement signaling can serve as a prognostic factor for clinical outcome in PAH.Conclusions: This study establishes immunoglobulin-driven dysregulated complement activation as a critical pathobiological mechanism regulating proinflammatory and pro-proliferative processes in the initiation of experimental hypoxic PH and demonstrates complement signaling as a critical determinant of clinical outcome in PAH.
Assuntos
Ativação do Complemento/imunologia , Fibroblastos/imunologia , Hipertensão Pulmonar/imunologia , Imunoglobulina G/imunologia , Remodelação Vascular/imunologia , Animais , Complemento C3/imunologia , Complemento C5/imunologia , Fator B do Complemento/imunologia , Via Alternativa do Complemento/imunologia , Modelos Animais de Doenças , Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Humanos , Hipertensão Pulmonar/etiologia , Hipóxia/complicações , Imunoglobulinas/imunologia , Inflamação , Camundongos , Camundongos Knockout , Prognóstico , Hipertensão Arterial Pulmonar/imunologia , RatosRESUMO
AbstractAim: The complement system is activated in acute kidney injury (AKI). Anti-C5 antibody targets the common terminal portion of the complement cascade that generate the terminal complex C5b-9 and has a renal-protective effect in paroxysmal nocturnal hemoglobinuria. However, the anti-C5 antibody's role in ischemia/reperfusion (I/R)-induced AKI has not been fully investigated. We therefore evaluated its effect on the pathophysiological cascade of I/R-induced AKI.Methods: Sprague-Dawley rats underwent unilateral right kidney nephrectomies with simultaneous clamping of the contralateral hilum for 60 min (ischemia), followed by reperfusion. In addition to a placebo, two treatment groups received either high or low doses of anti-C5 monoclonal antibody. After 48 h, the rats were euthanized, blood was drawn to evaluate systemic inflammation and to estimate glomerular filtration rate (GFR). The remaining kidney was removed for pathological evaluation and intra-renal complement activation.Results: I/R induced significant intra-renal complement activation and systemic inflammation compared with unilateral nephrectomy group. The anti-C5 antibody ameliorated the intra-renal complement activation (intra-renal C3 and C6), reduced systemic inflammation (C-reactive protein, and systemic C3), decreased intra-renal acute tubular necrosis damage and improved GFR (seen by the sensitive marker, serum cystatin C; 1.63 mg/L (I/R + placebo), 1.36 mg/L (I/R + low dose) and 1.21 mg/L (I/R + high dose), p = .08 and .03 compared with I/R + placebo).Conclusion: In I/R-induced AKI, the monoclonal anti-C5 complement factor ameliorates intra renal complement activation, decreases local and systemic inflammation and may improve GFR.
Assuntos
Injúria Renal Aguda/tratamento farmacológico , Anticorpos Monoclonais/farmacologia , Complemento C5/antagonistas & inibidores , Traumatismo por Reperfusão/complicações , Injúria Renal Aguda/imunologia , Injúria Renal Aguda/patologia , Animais , Anticorpos Monoclonais/uso terapêutico , Apoptose/efeitos dos fármacos , Apoptose/imunologia , Ativação do Complemento/efeitos dos fármacos , Ativação do Complemento/imunologia , Complemento C5/imunologia , Modelos Animais de Doenças , Taxa de Filtração Glomerular/efeitos dos fármacos , Humanos , Rim/efeitos dos fármacos , Rim/imunologia , Rim/patologia , Masculino , Ratos , Ratos Sprague-Dawley , Traumatismo por Reperfusão/imunologiaRESUMO
Over the last decade there has been an explosion in complement therapies; one-third of the drugs in the clinic or in development target C5 protein. Eculizumab, a monoclonal antibody (mAb) that binds C5 and blocks its cleavage by the convertase, is the current reference standard treatment for atypical haemolytic uraemic syndrome (aHUS) and paroxysmal nocturnal haemoglobinuria (PNH) and in clinical trials for many other diseases. Here we describe a panel of novel anti-C5 mAb, including mAb that, like Eculizumab, are efficient inhibitors of complement but, unlike Eculizumab, inhibit across species, including human, rat, rabbit and guinea pig. Several inhibitory anti-C5 mAb were identified and characterized for C5 binding and lytic inhibitory capacity in comparison to current therapeutic anti-C5 mAb; three clones, 4G2, 7D4 and 10B6, were selected and further characterized for ligand specificity and affinity and cross-species inhibitory activity. The mAb 10B6 was human-specific whereas mAb 4G2 and 7D4 efficiently inhibited lysis by human, rabbit and rat serum, and weakly inhibited guinea pig complement; 7D4 also weakly inhibited mouse complement in vitro The rat C5-cross-reactive mAb 4G2, when administered intraperitoneally in a rat model of myasthenia gravis, effectively blocked the disease and protected muscle endplates from destruction. To our knowledge this is the first report of an anti-C5 function blocking mAb that permits preclinical studies in rats.
Assuntos
Anticorpos Monoclonais Murinos , Complemento C5/imunologia , Miastenia Gravis , Animais , Anticorpos Monoclonais Murinos/imunologia , Anticorpos Monoclonais Murinos/farmacologia , Complemento C5/antagonistas & inibidores , Reações Cruzadas , Modelos Animais de Doenças , Cobaias , Humanos , Camundongos , Miastenia Gravis/tratamento farmacológico , Miastenia Gravis/imunologia , Miastenia Gravis/patologia , Coelhos , Ratos , Especificidade da EspécieRESUMO
IgA nephropathy (IgAN) is the most common form of primary glomerulonephritis worldwide and a common cause of end-stage renal disease. Evaluation of a kidney biopsy is necessary for diagnosis, with routine immunofluorescence microscopy revealing dominant or co-dominant IgA immunodeposits usually with complement C3 and sometimes IgG and/or IgM. IgA nephropathy reduces life expectancy by more than 10 years and leads to kidney failure in 20-40% of patients within 20 years of diagnosis. There is accumulating clinical, genetic, and biochemical evidence that complement plays an important role in the pathogenesis of IgA nephropathy. The presence of C3 differentiates the diagnosis of IgA nephropathy from the subclinical deposition of glomerular IgA. Markers for the activation of the alternative and mannan-binding lectin (MBL) pathways in renal-biopsy specimens are associated with disease activity and portend a worse renal outcome. Complement proteins in the circulation have also been evaluated in IgA nephropathy and found to be of prognostic value. Recently, genetic studies have identified IgA nephropathy-associated loci. Within these loci are genes encoding products involved in complement regulation and interaction with immune complexes. Put together, these data identify the complement cascade as a rational treatment target for this chronic kidney disease. Recent case reports on the successful use of humanized anti-C5 monoclonal antibody eculizumab are consistent with this hypothesis, but a better understanding of the role of complement in IgA nephropathy is needed to guide future therapeutic interventions.
Assuntos
Complemento C3/imunologia , Complemento C5/imunologia , Glomerulonefrite por IGA/imunologia , Imunoglobulina A/imunologia , Rim/imunologia , Animais , Glomerulonefrite/imunologia , Humanos , Insuficiência Renal Crônica/imunologiaRESUMO
Complement plays an important role in the pathogenesis of lupus nephritis (LN). With the emergence of therapeutic complement inhibition, there is a need to identify patients in whom complement-driven inflammation is a major cause of kidney injury in LN. Clinical and histopathological data were obtained retrospectively from 57 biopsies with class III, IV, and V LN. Biopsies were stained for complement components C9, C5b-9, C3c, and C3d and for the macrophage marker CD68. C9 and C5b-9 staining were highly correlated (r = 0.92 in the capillary wall). C5b-9 staining was detected in the mesangium and/or capillary wall of both active and chronic proliferative LN in all but one biopsy and in the capillary wall of class V LN in all biopsies. C5b-9 staining intensity in the tubular basement membrane correlated with markers of tubulointerstitial damage, and more intense capillary wall C5b-9 staining was significantly associated with nonresponse to conventional treatment. Glomerular C5b-9 staining intensity did not differ between active and chronic disease; in contrast, C3c and CD68 staining were associated with active disease. Evaluation of serial biopsies and comparison of staining in active and chronic LN demonstrated that C5b-9 staining persisted for months to years. These results suggest that C5b-9 staining is almost always present in LN, resolves slowly, and is not a reliable marker of ongoing glomerular C5 activation. This limits the utility of C5b-9 staining to identify patients who are most likely to benefit from C5 inhibition.
Assuntos
Ativação do Complemento , Complemento C5/imunologia , Complexo de Ataque à Membrana do Sistema Complemento/análise , Glomérulos Renais/patologia , Nefrite Lúpica/imunologia , Adolescente , Adulto , Idoso , Biomarcadores/análise , Biópsia , Complemento C5/antagonistas & inibidores , Complexo de Ataque à Membrana do Sistema Complemento/imunologia , Feminino , Humanos , Imunossupressores/farmacologia , Imunossupressores/uso terapêutico , Glomérulos Renais/imunologia , Nefrite Lúpica/tratamento farmacológico , Nefrite Lúpica/patologia , Masculino , Pessoa de Meia-Idade , Seleção de Pacientes , Reprodutibilidade dos Testes , Estudos Retrospectivos , Adulto JovemRESUMO
Purpose: Variants of complement factor genes, hypoxia and oxidative stress of the outer retina, and systemic hypertension affect the risk of age-related macular degeneration. Hypertension often results from the high intake of dietary salt that increases extracellular osmolarity. We determined the effects of extracellular hyperosmolarity, hypoxia, and oxidative stress on the expression of complement genes in cultured (dedifferentiated) human RPE cells and investigated the effects of C9 siRNA and C9 protein on RPE cells. Methods: Hyperosmolarity was induced by adding 100 mM NaCl or sucrose to the culture medium. Hypoxia was induced by culturing cells in 1% O2 or by adding the hypoxia mimetic CoCl2. Oxidative stress was induced by adding H2O2. Gene and protein expression levels were determined with real-time RT-PCR, western blot, and ELISA analyses. The expression of the nuclear factor of activated T cell 5 (NFAT5) and complement factor (C9) was knocked down with siRNA. Results: Extracellular hyperosmolarity, hypoxia, and oxidative stress strongly increased the transcription of the C9 gene, while the expression of the C3, C5, CFH, and CFB genes was moderately altered or not altered at all. Hyperosmolarity also induced a moderate increase in the cytosolic C9 protein level. The hyperosmotic C9 gene expression was reduced by inhibitors of the p38 MAPK, ERK1/2, JNK, and PI3K signal transduction pathways and of the transcription factors STAT3 and NFAT5. The hypoxic C9 gene expression was reduced by a STAT3 inhibitor. The knockdown of C9 with siRNA decreased the hypoxic vascular endothelial growth factor (VEGF) and NLRP3 gene expression, the hypoxic secretion of VEGF, and the hyperosmotic expression of the NLRP3 gene. Exogenous C9 protein inhibited the hyperosmotic expression of the C9 gene, the hypoxic and hyperosmotic VEGF gene expression, and the hyperosmotic expression of the NLRP3 gene. Both C9 siRNA and C9 protein inhibited inflammasome activation under hyperosmotic conditions, as indicated by the decrease in the cytosolic level of mature IL-1ß. Conclusions: The expression of the C9 gene in cultured RPE cells is highly induced by extracellular hyperosmolarity, hypoxia, and oxidative stress. The data may support the assumption that C9 gene expression may stimulate the expression of inflammatory (NLRP3) and angiogenic growth factors (VEGF) in RPE cells. Extracellular C9 protein may attenuate this effect, in part via negative regulation of the C9 mRNA level.
Assuntos
Cobalto/farmacologia , Complemento C9/genética , Células Epiteliais/efeitos dos fármacos , Peróxido de Hidrogênio/farmacologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Cloreto de Sódio/farmacologia , Fator A de Crescimento do Endotélio Vascular/genética , Hipóxia Celular/efeitos dos fármacos , Linhagem Celular , Complemento C3/genética , Complemento C3/imunologia , Complemento C5/genética , Complemento C5/imunologia , Complemento C9/antagonistas & inibidores , Complemento C9/imunologia , Fator B do Complemento/genética , Fator B do Complemento/imunologia , Fator H do Complemento/genética , Fator H do Complemento/imunologia , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Regulação da Expressão Gênica , Humanos , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/imunologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/imunologia , Concentração Osmolar , Pressão Osmótica/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Epitélio Pigmentado da Retina/citologia , Epitélio Pigmentado da Retina/efeitos dos fármacos , Epitélio Pigmentado da Retina/metabolismo , Fatores de Transcrição STAT/genética , Fatores de Transcrição STAT/imunologia , Transdução de Sinais , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Fatores de Transcrição/imunologia , Fator A de Crescimento do Endotélio Vascular/imunologiaRESUMO
The generation of high-quality protein crystals and the loss of phase information during an X-ray crystallography diffraction experiment represent the major bottlenecks in the determination of novel protein structures. A generic method for introducing Hg atoms into any crystal independent of the presence of free cysteines in the target protein could considerably facilitate the process of obtaining unbiased experimental phases. Nanobodies (single-domain antibodies) have recently been shown to promote the crystallization and structure determination of flexible proteins and complexes. To extend the usability of nanobodies for crystallographic work, variants of the Nb36 nanobody with a single free cysteine at one of four framework-residue positions were developed. These cysteines could be labelled with fluorophores or Hg. For one cysteine variant (Nb36-C85) two nanobody structures were experimentally phased using single-wavelength anomalous dispersion (SAD) and single isomorphous replacement with anomalous signal (SIRAS), taking advantage of radiation-induced changes in Cys-Hg bonding. Importantly, Hg labelling influenced neither the interaction of Nb36 with its antigen complement C5 nor its structure. The results suggest that Cys-Hg-labelled nanobodies may become efficient tools for obtaining de novo phase information during the structure determination of nanobody-protein complexes.
Assuntos
Cisteína/química , Mercúrio/química , Anticorpos de Domínio Único/química , Animais , Camelídeos Americanos , Complemento C5/imunologia , Cristalização , Cristalografia por Raios X , Humanos , Modelos Moleculares , Conformação Proteica , Anticorpos de Domínio Único/imunologiaRESUMO
BACKGROUND: C5 blockade by eculizumab prevents complement-mediated intravascular hemolysis in paroxysmal nocturnal hemoglobinuria (PNH). However, C3-bound PNH red blood cells (RBCs), arising in almost all treated patients, may undergo extravascular hemolysis reducing clinical benefits. Despite the uniform deficiency of CD55 and of CD59, there are always two distinct populations of PNH RBCs, with (C3+) and without (C3-) C3 binding. METHODS: To investigate this paradox, the phenomenon has been modeled in vitro by incubating RBCs from eculizumab untreated PNH patients with compatible sera containing eculizumab, and by assessing the C3 binding after activation of complement alternative pathway. RESULTS: When RBCs from untreated patients were exposed in vitro to activated complement in the context of C5-blockade, there was the prompt appearance of a distinct C3+ PNH RBC population whose size increased with time and also with the rate of complement activation. Eventually, all PNH RBCs become C3+ to the same extent, without differences between old and young (reticulocytes) PNH RBCs. CONCLUSIONS: This study indicates that the distinct (C3+ and C3-) PNH RBC populations are not intrinsically different; rather, they result from a stochastic all-or-nothing phenomenon linked to the time-dependent cumulative probability of each individual PNH red cell to be exposed to levels of complement activation able to trigger C3 binding. These findings may envision novel approaches to reduce C3 opsonization and the subsequent extravascular hemolysis in PNH patients on eculizumab.
Assuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Ativação do Complemento/efeitos dos fármacos , Complemento C3/imunologia , Eritrócitos/efeitos dos fármacos , Hemoglobinúria Paroxística/tratamento farmacológico , Hemólise/efeitos dos fármacos , Antígenos CD59/imunologia , Complemento C5/antagonistas & inibidores , Complemento C5/imunologia , Eritrócitos/imunologia , Eritrócitos/patologia , Hemoglobinúria Paroxística/imunologia , Hemoglobinúria Paroxística/patologia , Humanos , Processos EstocásticosRESUMO
As a crucial arm of innate immunity, the complement cascade (ComC) is involved both in mobilization of normal hematopoietic stem/progenitor cells (HSPCs) from bone marrow (BM) into peripheral blood and in their homing to BM. Despite the fact that ComC cleavage fragments alone do not chemoattract normal HSPCs, we found that leukemia cell lines as well as clonogenic blasts from chronic myeloid leukemia and acute myeloid leukemia patients respond robustly to C3 and C5 cleavage fragments by chemotaxis and increased adhesion. This finding was supported by the detection of C3a and C5a receptors in cells from human malignant hematopoietic cell lines and patient blasts at the mRNA (reverse transcriptase-polymerase chain reaction) and protein level (fluorescence-activated cell sorting), and by the demonstration that these receptors respond to stimulation by C3a and C5a by phosphorylation of p42/44 and p38 mitogen-activated protein kinases (MAPK), and protein kinase B (PKB/AKT). We also found that inducible heme oxygenase 1 (HO-1) is a negative regulator of ComC-mediated trafficking of leukemic cells, and that stimulation of leukemic cells by C3 or C5 cleavage fragments activates p38 MAPK, which downregulates HO-1 expression, rendering cells more mobile. We conclude that activation of the ComC in leukemia/lymphoma patients (for example, as a result of accompanying infections) enhances the motility of malignant cells and contributes to their spread in a p38 MAPK-HO-1-dependent manner. Therefore, inhibition of p38 MAPK or upregulation of HO-1 by small-molecule modulators would have a beneficial effect on ameliorating cell migration-mediated expansion of leukemia/lymphoma cells when the ComC becomes activated.
Assuntos
Ativação do Complemento/imunologia , Regulação Leucêmica da Expressão Gênica , Heme Oxigenase-1/genética , Leucemia/genética , Leucemia/imunologia , Animais , Adesão Celular/genética , Adesão Celular/imunologia , Linhagem Celular Tumoral , Movimento Celular/imunologia , Quimiotaxia/genética , Quimiotaxia/imunologia , Complemento C3/imunologia , Complemento C3/metabolismo , Complemento C5/imunologia , Complemento C5/metabolismo , Regulação para Baixo , Citometria de Fluxo , Técnicas de Inativação de Genes , Células-Tronco Hematopoéticas/metabolismo , Xenoenxertos , Humanos , Imunofenotipagem , Camundongos , Proteólise , RNA Interferente Pequeno/genética , Receptor da Anafilatoxina C5a/genética , Receptor da Anafilatoxina C5a/metabolismo , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico/metabolismo , Transdução de Sinais , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
PURPOSE OF REVIEW: The complement system represents one of the more primitive forms of innate immunity. It has increasingly been found to contribute to pathologies in the native and transplanted kidney. We provide a concise review of the physiology of the complement cascade, and discuss current and upcoming complement-based therapies. RECENT FINDINGS: Current agents in clinical use either bind to complement components directly or prevent complement from binding to antibodies affixed to the endothelial surface. These include C1 esterase inhibitors, anti-C5 mAbs, anti-CD20 mAbs, and proteasome inhibitors. Treatment continues to show efficacy in the atypical hemolytic uremic syndrome and antibody-mediated rejection. Promising agents not currently available include CCX168, TP10, AMY-101, factor D inhibitors, coversin, and compstatin. Several new trials are targeting complement inhibition to treat antineutrophilic cystoplasmic antibody (ANCA)-associated vasculitis, C3 glomerulopathy, thrombotic microangiopathy, and IgA nephropathy. New agents for the treatment of the atypical hemolytic uremic syndrome are also in development. SUMMARY: Complement-based therapies are being considered for targeted therapy in the atypical hemolytic uremic syndrome and antibody-mediated rejection, C3 glomerulopathy, and ANCA-associated vasculitis. A few agents are currently in use as orphan drugs. A number of other drugs are in clinical trials and, overall, are showing promising preliminary results.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Proteínas do Sistema Complemento/metabolismo , Compostos de Anilina/uso terapêutico , Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos/tratamento farmacológico , Antígenos CD20/imunologia , Síndrome Hemolítico-Urêmica Atípica/tratamento farmacológico , Ativação do Complemento/efeitos dos fármacos , Complemento C5/antagonistas & inibidores , Complemento C5/imunologia , Fator D do Complemento/antagonistas & inibidores , Glomerulonefrite por IGA/tratamento farmacológico , Humanos , Ácidos Nipecóticos/uso terapêutico , Peptídeos Cíclicos/uso terapêutico , Inibidores de Proteassoma/uso terapêutico , Receptores de Complemento/uso terapêutico , Microangiopatias Trombóticas/tratamento farmacológicoRESUMO
Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hemolytic anemia characterized by complement-mediated intravascular hemolysis that is effectively treated with eculizumab. However, treatment responses are reported heterogeneous with some patients presenting residual hemolysis and requiring RBC transfusions. Recent reports have shown that both extravascular hemolysis and incomplete C5 blockade can explain these suboptimal hematological responses. Here we have tested our eculizumab-treated PNH patients (n=12) for signs of hemolysis and assessed complement biomarkers. Patients were also genotyped for complement receptor 1 (CR1, CD35) and C5 polymorphisms and evaluated for free eculizumab in plasma. We report that 10 patients (83%) present parameters suggesting persistent hemolysis, although they did not require additional transfusions. Seven of them (58%) become direct Coombs-test positive as a consequence of treatment, including all patients carrying the low-expression CR1-L allele. CH50 and sC5b-9 assays demonstrate that the persistent low-level hemolysis identified in our treated patients is not a consequence of incomplete C5 blockade, supporting that this hemolysis, as has been suggested previously, results from the extravascular removal of C3 opsonized PNH erythrocytes. We also show that continuous alternative pathway activation in eculizumab-treated individuals carrying the CR1-L allele results in abnormally decreased levels of C3 in plasma that could, potentially, increase their susceptibility to bacterial infections. Finally, we encourage a routine evaluation of free eculizumab levels and terminal pathway activity to personalize eculizumab administration.
Assuntos
Proteínas do Sistema Complemento/imunologia , Hemoglobinúria Paroxística/sangue , Hemoglobinúria Paroxística/imunologia , Hemólise/imunologia , Anticorpos Monoclonais Humanizados/uso terapêutico , Criança , Pré-Escolar , Ativação do Complemento/efeitos dos fármacos , Ativação do Complemento/imunologia , Complemento C3/imunologia , Complemento C5/imunologia , Inativadores do Complemento/uso terapêutico , Citotoxicidade Imunológica , Feminino , Predisposição Genética para Doença , Variação Genética , Genótipo , Hemoglobinúria Paroxística/diagnóstico , Hemoglobinúria Paroxística/tratamento farmacológico , Humanos , Lactente , Masculino , Receptores de Complemento 3b/genética , Receptores de Complemento 3b/metabolismo , Resultado do TratamentoRESUMO
Endothelial cells (EC) play a central role in inflammation. E-selectin and ICAM-1 expression are essential for leukocyte recruitment and are good markers of EC activation. Most studies of EC activation are done in vitro using isolated mediators. The aim of the present study was to examine the relative importance of pattern recognition systems and downstream mediators in bacteria-induced EC activation in a physiological relevant human model, using EC incubated with whole blood. HUVEC were incubated with human whole blood. Escherichia coli- and Staphylococcus aureus-induced EC activation was measured by E-selectin and ICAM-1 expression using flow cytometry. The mAb 18D11 was used to neutralize CD14, and the lipid A analog eritoran was used to block TLR4/MD2. C5 cleavage was inhibited using eculizumab, and C5aR1 was blocked by an antagonist. Infliximab and canakinumab were used to neutralize TNF and IL-1ß. The EC were minimally activated when bacteria were incubated in serum, whereas a substantial EC activation was seen when the bacteria were incubated in whole blood. E. coli-induced activation was largely CD14-dependent, whereas S. aureus mainly caused a C5aR1-mediated response. Combined CD14 and C5 inhibition reduced E-selectin and ICAM-1 expression by 96 and 98% for E. coli and by 70 and 75% for S. aureus. Finally, the EC activation by both bacteria was completely abolished by combined inhibition of TNF and IL-1ß. E. coli and S. aureus activated EC in a CD14- and C5-dependent manner with subsequent leukocyte secretion of TNF and IL-1ß mediating the effect.