RESUMO
BACKGROUND: Homozygous CD59-deficient patients manifest with recurrent peripheral neuropathy resembling Guillain-Barré syndrome (GBS), hemolytic anemia and recurrent strokes. Variable mutations in CD59 leading to loss of function have been described and, overall, 17/18 of patients with any mutation presented with recurrent GBS. Here we determine the localization and possible role of membrane-bound complement regulators, including CD59, in the peripheral nervous systems (PNS) of mice and humans. METHODS: We examined the localization of membrane-bound complement regulators in the peripheral nerves of healthy humans and a CD59-deficient patient, as well as in wild-type (WT) and CD59a-deficient mice. Cross sections of teased sciatic nerves and myelinating dorsal root ganglia (DRG) neuron/Schwann cell cultures were examined by confocal and electron microscopy. RESULTS: We demonstrate that CD59a-deficient mice display normal peripheral nerve morphology but develop myelin abnormalities in older age. They normally express myelin protein zero (P0), ankyrin G (AnkG), Caspr, dystroglycan, and neurofascin. Immunolabeling of WT nerves using antibodies to CD59 and myelin basic protein (MBP), P0, and AnkG revealed that CD59 was localized along the internode but was absent from the nodes of Ranvier. CD59 was also detected in blood vessels within the nerve. Finally, we show that the nodes of Ranvier lack other complement-membrane regulatory proteins, including CD46, CD55, CD35, and CR1-related gene-y (Crry), rendering this area highly exposed to complement attack. CONCLUSION: The Nodes of Ranvier lack CD59 and are hence not protected from complement terminal attack. The myelin unit in human PNS is protected by CD59 and CD55, but not by CD46 or CD35. This renders the nodes and myelin in the PNS vulnerable to complement attack and demyelination in autoinflammatory Guillain-Barré syndrome, as seen in CD59 deficiency.
Assuntos
Síndrome de Guillain-Barré , Proteínas de Membrana , Camundongos , Humanos , Animais , Nós Neurofibrosos , Proteínas do Sistema Complemento , Antígenos CD59/genética , Antígenos CD55/genéticaRESUMO
OBJECTIVE: To investigate phenotypic and molecular characteristics of a consanguineous family with autosomal-recessive, polyarticular, juvenile isiopathic arthriris (JIA) with extra-articular manifestations, including renal amyloidosis and Crohn's disease, associated with a novel homozygous truncating variant in LACC1. METHODS: Whole exome sequencing (WES) or targeted Sanger verification were performed in 15 participants. LACC1 expression and cytokine array were analysed in patient-derived and CRISPR/Cas9-generated LACC1-knockout macrophages (MÏ). RESULTS: A homozygous truncating variant (p.Glu348Ter) in LACC1 was identified in three affected and one asymptomatic family member, and predicted harmful by causing premature stop of the LACC1 protein sequences, and by absence from ethnically-matched controls and public variation databases. Expression studies in patient-derived macrophages (MÏ) showed no endogenous p.Glu348Ter-LACC1 RNA transcription or protein expression, compatible with nonsense-mediated mRNA decay. WES analysis in the asymptomatic homozygous subject for p. Glu348Ter-LACC1 detected an exclusive heterozygous variant (p.Arg928Gln) in complement component C5. Further complement activity analysis suggested a protective role for the p.Arg928Gln-C5 variant as a phenotypic modifier of LACC1-associated disease. Finally, cytokine profile analysis indicated increased levels of pro-inflammatory cytokines in LACC1-disrupted as compared with wild-type MÏ. CONCLUSIONS: Our findings reinforce the role of LACC1 disruption in autosomal-recessive JIA, extend the clinical spectrum and intra-familial heterogeneity of the disease-associated phenotype, indicate a modulatory effect of complement factor C5 on phenotypic severity, and suggest an inhibitory role for wild-type LACC1 on pro-inflammatory pathways.
Assuntos
Artrite Juvenil/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Mutação com Perda de Função/genética , Adolescente , Adulto , Artrite Juvenil/patologia , Proteína 9 Associada à CRISPR , Sistemas CRISPR-Cas , Citocinas/sangue , Feminino , Citometria de Fluxo , Edição de Genes , Humanos , Immunoblotting , Masculino , Linhagem , Sequenciamento do Exoma , Adulto JovemRESUMO
OBJECTIVES: Loss of the complement inhibitor CD55 leads to a syndrome of early-onset protein-losing enteropathy (PLE), associated with intestinal lymphangiectasia and susceptibility to large-vein thrombosis. The in vitro and short-term treatment benefits of eculizumab (C5-inhibitor) therapy for CD55-deficiency have been previously demonstrated. Here we present the 18-months treatment outcomes for 3 CD55-deficiency patients with sustained therapeutic response. METHODS: Three CD55-deficiency patients received off-label eculizumab treatment. Clinical and laboratory treatment outcomes included frequency and consistency of bowl movements, weight, patient/parent reports of overall well-being, and serum albumin and total protein levels. Membrane attack complex deposition on leukocytes was tested by flow cytometry, before and during eculizumab treatment. RESULTS: Marked clinical improvement was noted in all 3 patients with resolution of PLE manifestations, that is, diarrhea, edema, malabsorption, overall well-being, growth, and quality of life. In correlation with the clinical observations, we observed progress in all laboratory outcome parameters, including increase in albumin and total protein levels, and up to 80% reduction in membrane attack complex deposition on leukocytes (P < 0.001). The progress persisted over 18 months of treatment without any severe adverse events. CONCLUSIONS: CD55-deficiency patients present with early-onset diarrhea, edema, severe hypoalbuminemia, abdominal pain, and malnutrition. Targeted therapy with the terminal complement inhibitor eculizumab has positive clinical and laboratory outcomes in PLE related to CD55 loss-of-function mutations, previously a life-threatening condition. Our results demonstrate the potential of genetic diagnosis to guide tailored treatment, and underscore the significant role of the complement system in the intestine.
Assuntos
Anticorpos Monoclonais Humanizados/administração & dosagem , Antígenos CD55/deficiência , Inativadores do Complemento/administração & dosagem , Enteropatias Perdedoras de Proteínas/tratamento farmacológico , Adulto , Criança , Pré-Escolar , Ensaios de Uso Compassivo , Relação Dose-Resposta a Droga , Esquema de Medicação , Seguimentos , Humanos , Infusões Intravenosas , Linfangiectasia Intestinal/complicações , Linfangiectasia Intestinal/patologia , Uso Off-Label , Estudos Prospectivos , Enteropatias Perdedoras de Proteínas/etiologia , Qualidade de Vida , Indução de RemissãoRESUMO
Sepsis has no proven pharmacologic treatment other than appropriate antibiotic agents, fluids, vasopressors as needed, and possibly corticosteroids. It is generally initiated mainly by the simultaneous recognition by various components of the innate immune system of either pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs). In the current study, we employed the murine cecal ligation and puncture (CLP) model for sepsis to evaluate the effect of post-CLP infusion of apoptotic cells (Allocetra-OTS) on a CLP severe sepsis model. Cardiovascular evaluation, acute kidney injury (AKI), acute liver injury (ALI), and hematological and metabolic function were evaluated. Cytokine and chemokine profiles were measured by Multiplex ELISA and mitochondrial function, and glycolysis by Seahorse. The Murine Sepsis Score (MSS) was used for disease severity definition. CLP mice had low blood pressure, poor cardiac output, and lung dysfunction, as well as AKI, ALI, and thrombocytopenia, which correlated with the MSS and corresponded to a cytokine/chemokine storm. Apoptotic cell administration markedly improved the cytokine and chemokine storm and restored the impaired mitochondrial and glycolytic function in white blood cells leading to increased survival, from 6 to 60% (P < 0.0001), together with a significant improvement in organ dysfunction. We conclude that the deleterious immune response in CLP-induced sepsis can be successfully modified by apoptotic cell infusion.
Assuntos
Síndrome da Liberação de Citocina/complicações , Sepse/genética , Animais , Apoptose , Modelos Animais de Doenças , Masculino , Camundongos , Sepse/patologiaRESUMO
BACKGROUND: Thrombosis is the prognostic factor with the greatest effect on survival in patients with paroxysmal nocturnal hemoglobinuria (PNH), who lack dozens of membrane surface proteins. We recently described a primary homozygous Cys89Tyr congenital nonfunctioning CD59 in humans with clinical manifestation in infancy, associated with chronic hemolysis, recurrent strokes, and relapsing peripheral demyelinating neuropathy. Here we investigated hypercoagulability mechanisms characterizing the syndrome. METHODS: Membrane attack complex (MAC) deposition (anti-SC5b-9) and free hemoglobin (colorimetric assay) were assessed. Platelet activation was identified (anti-CD61, anti-CD62P), and microparticles (MPs) of 0.5-0.9⯵m, were characterized (Annexin V, anti-human GlyA, anti-CD15, anti-CD14, anti-CD61). Platelet-monocyte aggregation was assessed with FlowSight. FINDINGS: 2/7 patients (29%) with homozygosity for Cys89Tyr and 6/12 (50%) with any of four described CD59 mutations had recurrent strokes. In plasma samples from four patients carrying identical mutations, MAC deposition was increased on RBCs (pâ¯<â¯0.0003), neutrophils (pâ¯<â¯0.009), and platelets (pâ¯<â¯0.0003). Free-plasma hemoglobin levels were abnormally high, up to 100â¯mg/dl. Patients with CD59 mutation had RBC-derived MP levels 9-fold higher than those in healthy controls (pâ¯<â¯0.01), and 2-2.5 fold higher than PNH patients (pâ¯<â¯0.09). Leukocyte-activated platelet aggregation was increased (pâ¯<â¯0.0062). Loss of CD59 was shown in the endothelium of these patients. INTERPRETATION: Nonfunctioning CD59 is a major risk factor for stroke and hypercoagulability. Uncontrolled hemolysis causes massive MP release and endothelial heme damage. MAC attack on unprotected endothelium and platelet activation and aggregation with leukocytes mediate additional mechanisms leading to vascular occlusion. It is suggested that CD59 loss represents a major arterial prothrombotic factor in PNH and additional diseases.
Assuntos
Antígenos CD59/genética , Complexo de Ataque à Membrana do Sistema Complemento/genética , Trombose/genética , Feminino , Humanos , Masculino , Mutação , Trombose/patologiaRESUMO
OBJECTIVE: To characterize all 4 mutations described for CD59 congenital deficiency. METHODS: The 4 mutations, p.Cys64Tyr, p.Asp24Val, p.Asp24Valfs*, and p.Ala16Alafs*, were described in 13 individuals with CD59 malfunction. All 13 presented with recurrent Guillain-Barré syndrome or chronic inflammatory demyelinating polyneuropathy, recurrent strokes, and chronic hemolysis. Here, we track the molecular consequences of the 4 mutations and their effects on CD59 expression, localization, glycosylation, degradation, secretion, and function. Mutants were cloned and inserted into plasmids to analyze their expression, localization, and functionality. RESULTS: Immunolabeling of myc-tagged wild-type (WT) and mutant CD59 proteins revealed cell surface expression of p.Cys64Tyr and p.Asp24Val detected with the myc antibody, but no labeling by anti-CD59 antibodies. In contrast, frameshift mutants p.Asp24Valfs* and p.Ala16Alafs* were detected only intracellularly and did not reach the cell surface. Western blot analysis showed normal glycosylation but mutant-specific secretion patterns. All mutants significantly increased MAC-dependent cell lysis compared with WT. In contrast to CD59 knockout mice previously used to characterize phenotypic effects of CD59 perturbation, all 4 hCD59 mutations generate CD59 proteins that are expressed and may function intracellularly (4) or on the cell membrane (2). None of the 4 CD59 mutants are detected by known anti-CD59 antibodies, including the 2 variants present on the cell membrane. None of the 4 inhibits membrane attack complex (MAC) formation. CONCLUSIONS: All 4 mutants generate nonfunctional CD59, 2 are expressed as cell surface proteins that may function in non-MAC-related interactions and 2 are expressed only intracellularly. Distinct secretion of soluble CD59 may have also a role in disease pathogenesis.
RESUMO
Neurological symptoms of patients with p.Cys89Tyr mutation in the CD59 gene include recurrent peripheral neuropathy resembling Guillain-Barré syndrome, characterized by sensory-motor demyelinating neuropathy with secondary axonal damage and moderate enhancement of the nerve roots on spine MRI, together with recurrent strokes and retinal involvement. Three additional mutations in CD59, leading to loss of function, have been described, and overall, 12/12 (100%) of patients with any mutation presented with neurological symptoms; 11/12 (92%) patients presented with recurrent peripheral neuropathy, 6/12 (50%) with recurrent strokes, and 1/12 (8%) with retinal involvement. We review the possible thrombophilic profile associated with the mutations. In these patients, excessive intravascular hemolysis saturates scavenger mechanisms resulting in free hemoglobin in plasma that irreversibly reacts with nitric oxide to form nitrate and methemoglobin, leading to arterial thrombosis. CD59 loss of function is also one of the major thrombophilic mechanisms in patients with paroxysmal nocturnal hemoglobinuria. We then describe the relationship with demyelination. The lack of CD59 allows uncontrolled complement amplification following low-level spontaneous-, viral-, or post viral-induced complement activation, resulting in severe demyelination in the peripheral nervous system. It is interesting, and certainly encouraging, that after 3 years, following 4 patients with Cys89Tyr mutations who are treated with eculizumab, no strokes occurred and non-permanent neurological insults underwent resolution without any new neurological exacerbations.
Assuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Antígenos CD59/genética , Doenças Desmielinizantes/tratamento farmacológico , Mutação , Acidente Vascular Cerebral/tratamento farmacológico , Ativação do Complemento/efeitos dos fármacos , Inativadores do Complemento/uso terapêutico , Doenças Desmielinizantes/genética , Humanos , Acidente Vascular Cerebral/genética , Resultado do TratamentoRESUMO
Cysteine catabolism presents cells with a double-edged sword. On the one hand, cysteine degradation provides cells with essential molecules such as taurine and sulfide. The formation of sulfide in cells is thought to regulate important and diverse physiological processes including blood circulation, synaptic activity and inflammation. On the other hand, the catabolism of cysteine by gut microbiota can release high levels of sulfide that may underlie the development or relapse of ulcerative colitis, an inflammatory bowel disease affecting millions of people worldwide. Here, we have used the nematode C. elegans to explore how cells tolerate high levels of sulfide produced by cysteine degradation in bacteria. We have identified mutations in genes coding for thioredoxin family proteins, mitochondrial proteins, and collagens that confer tolerance to sulfide toxicity. Exposure to sulfide induces the unfolded protein response in the endoplasmic reticulum and mitochondria. Moreover, our results suggest that sulfide toxicity is mediated by reactive oxygen species (ROS). Indeed, pre-treatment of worms with antioxidants increases their tolerance to sulfide toxicity. Intriguingly, sub-toxic levels of the superoxide generator paraquat can also increase the tolerance of worms to sulfide. Therefore, it appears that activation of ROS detoxification pathway prior to the exposure to sulfide, can increase the tolerance to sulfide toxicity. Our results suggest that these detoxification pathways are mediated by the hypoxia inducible factor HIF-1. Finally, we show that sulfide resistance varies among wild C. elegans and other nematode species, suggesting that tolerance to sulfide was naturally selected in certain habitats.