RESUMO
Following viral infection, T-cells are crucial for an effective immune response to intracellular pathogens, including respiratory viruses. During the COVID-19 pandemic, diverse assays were required in pre-clinical trials to evaluate the immune response following vaccination against SARS-CoV-2 and assess the response following exposure to the virus. To assess the nature and potency of the cellular response to infection or vaccination, a reliable and specific activity assay was needed. A cellular activity assay based on the presentation of short peptides (epitopes) allows the identification of T cell epitopes displayed on different alleles of the MHC, shedding light on the strength of the immune response towards antigens and aiding in antigen design for vaccination. In this report, we describe two approaches for scanning T cell epitopes on the surface glycoprotein of the SARS-CoV-2 (spike), which is utilized for attachment and entry and serves as an antigen in many vaccine candidates. We demonstrate that epitope scanning is feasible using peptide libraries or computational scanning combined with a cellular activity assay. Our scans identified four CD8 T cell epitopes, including one novel undescribed epitope. These epitopes enabled us to establish a reliable T-cell response assay, which was examined and used in various experimental mouse models for SARS-CoV-2 infection and vaccination. These approaches could potentially aid in future antigen design for vaccination and establish cellular activity assays against uncharacterized antigens of emerging pathogens.
RESUMO
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the COVID-19 pandemic. Currently, as dangerous mutations emerge, there is an increased demand for specific treatments for SARS-CoV-2 infected patients. The spike glycoprotein on the virus envelope binds to the angiotensin converting enzyme 2 (ACE2) on host cells through its receptor binding domain (RBD) to mediate virus entry. Thus, blocking this interaction may inhibit viral entry and consequently stop infection. Here, we generated fusion proteins composed of the extracellular portions of ACE2 and RBD fused to the Fc portion of human IgG1 (ACE2-Ig and RBD-Ig, respectively). We demonstrate that ACE2-Ig is enzymatically active and that it can be recognized by the SARS-CoV-2 RBD, independently of its enzymatic activity. We further show that RBD-Ig efficiently inhibits in-vivo SARS-CoV-2 infection better than ACE2-Ig. Mechanistically, we show that anti-spike antibody generation, ACE2 enzymatic activity, and ACE2 surface expression were not affected by RBD-Ig. Finally, we show that RBD-Ig is more efficient than ACE2-Ig at neutralizing high virus titers. We thus propose that RBD-Ig physically blocks virus infection by binding to ACE2 and that RBD-Ig should be used for the treatment of SARS-CoV-2-infected patients.
Assuntos
Enzima de Conversão de Angiotensina 2/antagonistas & inibidores , Fragmentos Fc das Imunoglobulinas/metabolismo , Imunoglobulina G/metabolismo , Domínios Proteicos , Proteínas Recombinantes de Fusão/metabolismo , SARS-CoV-2/metabolismo , Enzima de Conversão de Angiotensina 2/metabolismo , Animais , Sítios de Ligação , Sítios de Ligação de Anticorpos , COVID-19/prevenção & controle , Chlorocebus aethiops , Feminino , Células HEK293 , Humanos , Fragmentos Fc das Imunoglobulinas/uso terapêutico , Imunoglobulina G/uso terapêutico , Camundongos Transgênicos , Testes de Neutralização , Ligação Proteica , Proteínas Recombinantes de Fusão/uso terapêutico , SARS-CoV-2/efeitos dos fármacos , Células VeroRESUMO
The ongoing COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a major threat to global health. Vaccines are ideal solutions to prevent infection, but treatments are also needed for those who have contracted the virus to limit negative outcomes, when vaccines are not applicable. Viruses must cross host cell membranes during their life cycle, creating a dependency on processes involving membrane dynamics. Thus, in this study, we examined whether the synthetic machinery for glycosphingolipids, biologically active components of cell membranes, can serve as a therapeutic target to combat SARS-CoV-2. We examined the antiviral effect of two specific inhibitors of glucosylceramide synthase (GCS): (i) Genz-123346, an analogue of the United States Food and Drug Administration-approved drug Cerdelga and (ii) GENZ-667161, an analogue of venglustat, which is currently under phase III clinical trials. We found that both GCS inhibitors inhibit replication of SARS-CoV-2. Moreover, these inhibitors also disrupt replication of influenza virus A/PR/8/34 (H1N1). Our data imply that synthesis of glycosphingolipids is necessary to support viral life cycles and suggest that GCS inhibitors should be further explored as antiviral therapies.
Assuntos
Antivirais/farmacologia , Carbamatos/farmacologia , Dioxanos/farmacologia , Glucosiltransferases/antagonistas & inibidores , Glicoesfingolipídeos/antagonistas & inibidores , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Pirrolidinas/farmacologia , Quinuclidinas/farmacologia , SARS-CoV-2/efeitos dos fármacos , Animais , Antivirais/síntese química , COVID-19/enzimologia , COVID-19/virologia , Carbamatos/síntese química , Membrana Celular/efeitos dos fármacos , Membrana Celular/enzimologia , Membrana Celular/virologia , Chlorocebus aethiops , Ensaios Clínicos Fase III como Assunto , Dioxanos/síntese química , Cães , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Glicoesfingolipídeos/biossíntese , Interações Hospedeiro-Patógeno/genética , Humanos , Vírus da Influenza A Subtipo H1N1/crescimento & desenvolvimento , Vírus da Influenza A Subtipo H1N1/metabolismo , Influenza Humana/tratamento farmacológico , Influenza Humana/enzimologia , Influenza Humana/virologia , Células Madin Darby de Rim Canino , Pirrolidinas/síntese química , Quinuclidinas/síntese química , SARS-CoV-2/crescimento & desenvolvimento , SARS-CoV-2/metabolismo , Transdução de Sinais , Células Vero , Replicação Viral/efeitos dos fármacos , Tratamento Farmacológico da COVID-19RESUMO
BACKGROUND: Although coronavirus disease 2019 (COVID-19) causes significan t morbidity, mainly from pulmonary involvement, extrapulmonary symptoms are also major componen ts of the disease. Kidney disease, usually presenting as AKI, is particularly severe among patients with COVID-19. It is unknown, however, whether such injury results from direct kidney infection with COVID-19's causative virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), or from indirect mechanisms. METHODS: Using ex vivo cell models, we sought to analyze SARS-CoV-2 interactions with kidney tubular cells and assess direct tubular injury. These models comprised primary human kidney epithelial cells (derived from nephrectomies) and grown as either proliferating monolayers or quiescent three-dimensional kidney spheroids. RESULTS: We demonstrated that viral entry molecules and high baseline levels of type 1 IFN-related molecules were present in monolayers and kidney spheroids. Although both models support viral infection and replication, they did not exhibit a cytopathic effect and cell death, outcomes that were strongly present in SARS-CoV-2-infected controls (African green monkey kidney clone E6 [Vero E6] cultures). A comparison of monolayer and spheroid cultures demonstrated higher infectivity and replication of SARS-CoV-2 in actively proliferating monolayers, although the spheroid cultures exhibited high er levels of ACE2. Monolayers exhibited elevation of some tubular injury molecules-including molecules related to fibrosis (COL1A1 and STAT6) and dedifferentiation (SNAI2)-and a loss of cell identity, evident by reduction in megalin (LRP2). The three-dimensional spheroids were less prone to such injury. CONCLUSIONS: SARS-CoV-2 can infect kidney cells without a cytopathic effect. AKI-induced cellular proliferation may potentially intensify infectivity and tubular damage by SARS-CoV-2, suggesting that early intervention in AKI is warranted to help minimize kidney infection.
Assuntos
Injúria Renal Aguda/etiologia , Injúria Renal Aguda/virologia , COVID-19/complicações , SARS-CoV-2/patogenicidade , Esferoides Celulares/virologia , Animais , Células Cultivadas , Chlorocebus aethiops , Estudos de Coortes , Efeito Citopatogênico Viral , Células Epiteliais/patologia , Células Epiteliais/virologia , Interações entre Hospedeiro e Microrganismos , Humanos , Interferon Tipo I/metabolismo , Rim/imunologia , Rim/patologia , Rim/virologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Modelos Biológicos , Pandemias , Receptores Virais/metabolismo , Estudos Retrospectivos , SARS-CoV-2/fisiologia , Esferoides Celulares/patologia , Células Vero , Replicação ViralRESUMO
Most lysosomal storage diseases (LSDs) have a significant neurological component, including types 2 and 3 Gaucher disease (neuronal forms of Gaucher disease; nGD). No therapies are currently available for nGD since the recombinant enzymes used in the systemic form of Gaucher disease do not cross the blood-brain barrier (BBB). However, a number of promising approaches are currently being tested, including substrate reduction therapy (SRT), in which partial inhibition of the synthesis of the glycosphingolipids (GSLs) that accumulate in nGD lowers their accumulation. We now induce nGD in mice by injection with conduritol B-epoxide (CBE), an irreversible inhibitor of acid beta-glucosidase (GCase), the enzyme defective in nGD, with or without co-injection with Genz-667161, a prototype for SRT which crosses the BBB. Significant neuropathology, and a reduction in lifespan, was observed upon CBE injection, and this was largely reversed by co-injection with Genz-667161, along with a reduction in glucosylceramide and glucosylsphingosine levels. Analysis of gene expression by RNAseq revealed that Genz-667161 largely reversed the changes in genes and pathways that were differentially expressed upon CBE injection, specifically pathways of GSL metabolism, lipoproteins and other lipid metabolic pathways, lipid droplets, astrocyte activation, neuronal function, and to some extent, neuroinflammation. Together, this demonstrates the efficacy of SRT to reverse the effects of substrate accumulation on pathological components and pathways in nGD brain.
Assuntos
Modelos Animais de Doenças , Doença de Gaucher/metabolismo , Doença de Gaucher/patologia , Glucosilceramidase/antagonistas & inibidores , Glicoesfingolipídeos/antagonistas & inibidores , Animais , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/metabolismo , Barreira Hematoencefálica/patologia , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Feminino , Doença de Gaucher/tratamento farmacológico , Glucosilceramidase/metabolismo , Glicoesfingolipídeos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Especificidade por Substrato/efeitos dos fármacos , Especificidade por Substrato/fisiologiaRESUMO
Great interest has been shown in understanding the pathology of Gaucher disease (GD) due to the recently discovered genetic relationship with Parkinson's disease. For such studies, suitable animal models of GD are required. Chemical induction of GD by inhibition of acid ß-glucosidase (GCase) using the irreversible inhibitor conduritol B-epoxide (CBE) is particularly attractive, although few systematic studies examining the effect of CBE on the development of symptoms associated with neurological forms of GD have been performed. We now demonstrate a correlation between the amount of CBE injected into mice and levels of accumulation of the GD substrates, glucosylceramide and glucosylsphingosine, and show that disease pathology, indicated by altered levels of pathological markers, depends on both the levels of accumulated lipids and the time at which their accumulation begins. Gene array analysis shows a remarkable similarity in the gene expression profiles of CBE-treated mice and a genetic GD mouse model, the Gba(flox/flox) ;nestin-Cre mouse, with 120 of the 144 genes up-regulated in CBE-treated mice also up-regulated in Gba(flox/flox) ;nestin-Cre mice. We also demonstrate that various aspects of neuropathology and some behavioural abnormalities can be arrested upon cessation of CBE treatment during a specific time window. Together, our data demonstrate that injection of mice with CBE provides a rapid and relatively easy way to induce symptoms typical of neuronal forms of GD. This is particularly useful when examining the role of specific biochemical pathways in GD pathology, since CBE can be injected into mice defective in components of putative pathological pathways, alleviating the need for time-consuming crossing of mice. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Assuntos
Doença de Gaucher/patologia , Animais , Modelos Animais de Doenças , Doença de Gaucher/induzido quimicamente , Doença de Gaucher/genética , Perfilação da Expressão Gênica , Inositol/análogos & derivados , CamundongosRESUMO
Gaucher disease has recently received wide attention due to the unexpected discovery that it is a genetic risk factor for Parkinson's disease. Gaucher disease is caused by the defective activity of the lysosomal enzyme, glucocerebrosidase (GCase; GBA1), resulting in intracellular accumulation of the glycosphingolipids, glucosylceramide and psychosine. The rare neuronopathic forms of GD (nGD) are characterized by profound neurological impairment and neuronal cell death. We have previously described the progression of neuropathological changes in a mouse model of nGD. We now examine the relationship between glycosphingolipid accumulation and initiation of pathology at two pre-symptomatic stages of the disease in four different brain areas which display differential degrees of susceptibility to GCase deficiency. Liquid chromatography electrospray ionization tandem mass spectrometry demonstrated glucosylceramide and psychosine accumulation in nGD brains prior to the appearance of neuroinflammation, although only glucosylceramide accumulation correlated with neuroinflammation and neuron loss. Levels of other sphingolipids, including the pro-apoptotic lipid, ceramide, were mostly unaltered. Transmission electron microscopy revealed that glucosylceramide accumulation occurs in neurons, mostly in the form of membrane-delimited pseudo-tubules located near the nucleus. Highly disrupted glucosylceramide-storing cells, which are likely degenerating neurons containing massive inclusions, numerous autophagosomes and unique ultrastructural features, were also observed. Together, our results indicate that a certain level of neuronal glucosylceramide storage is required to trigger neuropathological changes in affected brain areas, while other brain areas containing similar glucosylceramide levels are unaltered, presumably because of intrinsic differences in neuronal properties, or in the neuronal environment, between various brain regions.
Assuntos
Doença de Gaucher/metabolismo , Glucosilceramidas/metabolismo , Degeneração Neural/metabolismo , Neurônios/metabolismo , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Doença de Gaucher/patologia , Glucosilceramidase/deficiência , Glucosilceramidase/genética , Humanos , Lactosilceramidas/metabolismo , Camundongos , Camundongos Knockout , Neurônios/patologia , Psicosina/metabolismo , Esfingomielinas/metabolismoRESUMO
BACKGROUND: Neuroinflammation is a key phenomenon in the pathogenesis of many neurodegenerative diseases. Understanding the mechanisms by which brain inflammation is engaged and delineating the key players in the immune response and their contribution to brain pathology is of great importance for the identification of novel therapeutic targets for these devastating diseases. Gaucher disease, the most common lysosomal storage disease, is caused by mutations in the GBA1 gene and is a significant risk factor for Parkinson's disease; in some forms of Gaucher disease, neuroinflammation is observed. METHODS: An unbiased gene profile analysis was performed on a severely affected brain area of a neurological form of a Gaucher disease mouse at a pre-symptomatic stage; the mouse used for this study, the Gba (flox/flox); nestin-Cre mouse, was engineered such that GBA1 deficiency is restricted to cells of neuronal lineage, i.e., neurons and macroglia. RESULTS: The 10 most up-regulated genes in the ventral posteromedial/posterolateral region of the thalamus were inflammatory genes, with the gene expression signature significantly enriched in interferon signaling genes. Interferon ß levels were elevated in neurons, and interferon-stimulated genes were elevated mainly in microglia. Interferon signaling pathways were elevated to a small extent in the brain of another lysosomal storage disease mouse model, Krabbe disease, but not in Niemann-Pick C or Sandhoff mouse brain. Ablation of the type I interferon receptor attenuated neuroinflammation but had no effect on GD mouse viability. CONCLUSIONS: Our results imply that the type I interferon response is involved in the development of nGD pathology, and possibly in other lysosomal storage diseases in which simple glycosphingolipids accumulate, and support the notion that interferon signaling pathways play a vital role in the sterile inflammation that often occurs during chronic neurodegenerative diseases in which neuroinflammation is present.
Assuntos
Encéfalo/metabolismo , Encéfalo/patologia , Doença de Gaucher , Interferon Tipo I/metabolismo , Neurônios/metabolismo , Regulação para Cima/genética , Animais , Receptor 1 de Quimiocina CX3C , Encefalite/etiologia , Encefalite/metabolismo , Encefalite/patologia , Doença de Gaucher/complicações , Doença de Gaucher/genética , Doença de Gaucher/metabolismo , Perfilação da Expressão Gênica , Glucosilceramidase/deficiência , Glucosilceramidase/genética , Glicoesfingolipídeos/metabolismo , Humanos , Recém-Nascido , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas do Tecido Nervoso/metabolismo , Nestina/genética , Nestina/metabolismo , Neurônios/patologia , Receptor de Interferon alfa e beta/deficiência , Receptor de Interferon alfa e beta/genética , Receptores de Quimiocinas/genética , Receptores de Quimiocinas/metabolismo , Transdução de Sinais/genéticaRESUMO
Lysosomal storage diseases (LSDs) are mainly caused by the defective activity of lysosomal hydrolases. A sub-class of LSDs are the sphingolipidoses, in which sphingolipids accumulate intra-cellularly. We here discuss the role of innate immunity in the sphingolipidoses, and compare the pathways of activation in two classical sphingolipidoses, namely Gaucher disease and Sandhoff disease, and in Niemann-Pick C disease, in which the main storage material is cholesterol but sphingolipids also accumulate. We discuss the mechanisms leading to neuroinflammation, and the different pathways of neuroinflammation in the different diseases, and suggest that intervention in these pathways may be a useful therapeutic approach to address these devastating human diseases.
Assuntos
Encéfalo/imunologia , Imunidade Inata/imunologia , Doenças por Armazenamento dos Lisossomos/imunologia , Esfingolipidoses/imunologia , Animais , Doença de Gaucher/imunologia , Humanos , Doença de Niemann-Pick Tipo C/imunologiaRESUMO
Artificial antigen-presenting cells (aAPCs) are currently used to manufacture T cells for adoptive therapy in cancer treatment, but a readily tunable and modular system can enable both rapid T cell expansion and control over T cell phenotype. Here, it is shown that microgels with tailored surface biochemical properties can serve as aAPCs to mediate T cell activation and expansion. Surface functionalization of microgels is achieved via layer-by-layer coating using oppositely charged polymers, forming a thin but dense polymer layer on the surface. This facile and versatile approach is compatible with a variety of coating polymers and allows efficient and flexible surface-specific conjugation of defined peptides or proteins. The authors demonstrate that tethering appropriate stimulatory ligands on the microgel surface efficiently activates T cells for polyclonal and antigen-specific expansion. The expansion, phenotype, and functional outcome of primary mouse and human T cells can be regulated by modulating the concentration, ratio, and distribution of stimulatory ligands presented on microgel surfaces as well as the stiffness and viscoelasticity of the microgels.
Assuntos
Células Apresentadoras de Antígenos , Microgéis , Propriedades de Superfície , Linfócitos T , Animais , Células Apresentadoras de Antígenos/imunologia , Linfócitos T/citologia , Linfócitos T/metabolismo , Humanos , Microgéis/química , Camundongos , Proliferação de Células/efeitos dos fármacos , Ativação Linfocitária , Polímeros/químicaRESUMO
Gaucher disease (GD), the most common lysosomal storage disorder, is caused by a deficiency in the lysosomal enzyme glucocerebrosidase (GlcCerase), which results in intracellular accumulation of glucosylceramide (GlcCer). The rare neuronopathic forms of GD are characterized by profound neurological impairment and neuronal cell death, but little is known about the neuropathological changes that underlie these events. We now systematically examine the onset and progression of various neuropathological changes (including microglial activation, astrogliosis and neuron loss) in a mouse model of neuronopathic GD, and document the brain areas that are first affected, which may reflect vulnerability of these areas to GlcCerase deficiency. We also identify neuropathological changes in several brain areas and pathways, such as the substantia nigra reticulata, reticulotegmental nucleus of the pons, cochlear nucleus and the somatosensory system, which could be responsible for some of the neurological manifestations of the human disease. In addition, we establish that microglial activation and astrogliosis are spatially and temporally correlated with selective neuron loss.
Assuntos
Encéfalo/patologia , Encéfalo/fisiopatologia , Modelos Animais de Doenças , Doença de Gaucher/patologia , Doença de Gaucher/fisiopatologia , Inflamação/fisiopatologia , Neurônios/patologia , Animais , Encéfalo/enzimologia , Morte Celular , Doença de Gaucher/enzimologia , Doença de Gaucher/genética , Glucosilceramidase/genética , Glucosilceramidase/metabolismo , Humanos , Inflamação/enzimologia , Inflamação/genética , Inflamação/patologia , Camundongos , Camundongos Mutantes , Neurônios/enzimologiaRESUMO
Gaucher's disease, the most common lysosomal storage disorder, is caused by the defective activity of glucocerebrosidase, the lysosomal hydrolase that degrades glucosylceramide. The neuronopathic forms of Gaucher's disease are characterized by severe neuronal loss, astrocytosis and microglial proliferation, but the cellular and molecular pathways causing these changes are not known. In the current study, we delineate the role of neuroinflammation in the pathogenesis of neuronopathic Gaucher's disease and show significant changes in levels of inflammatory mediators in the brain of a neuronopathic Gaucher's disease mouse model. Levels of messenger RNA expression of interleukin -1ß, tumour necrosis factor-α, tumour necrosis factor-α receptor, macrophage colony-stimulating factor and transforming growth factor-ß were elevated by up to â¼30-fold, with the time-course of the increase correlating with the progression of disease severity. The most significant elevation was detected for the chemokines CCL2, CCL3 and CCL5. Blood-brain barrier disruption was also evident in mice with neuronopathic Gaucher's disease. Finally, extensive elevation of nitrotyrosine, a hallmark of peroxynitrite (ONOO(-)) formation, was observed, consistent with oxidative damage caused by macrophage/microglia activation. Together, our results suggest a cytotoxic role for activated microglia in neuronopathic Gaucher's disease. We suggest that once a critical threshold of glucosylceramide storage is reached in neurons, a signalling cascade is triggered that activates microglia, which in turn releases inflammatory cytokines that amplify the inflammatory response, contributing to neuronal death.
Assuntos
Encefalite/etiologia , Doença de Gaucher/complicações , Neurônios/patologia , Animais , Animais Recém-Nascidos , Anti-Inflamatórios não Esteroides/uso terapêutico , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Barreira Hematoencefálica/fisiopatologia , Proteínas de Ligação ao Cálcio/metabolismo , Morte Celular/efeitos dos fármacos , Citocinas/genética , Citocinas/metabolismo , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Encefalite/diagnóstico , Encefalite/tratamento farmacológico , Encefalite/patologia , Células Endoteliais/patologia , Ensaio de Imunoadsorção Enzimática/métodos , Doença de Gaucher/tratamento farmacológico , Doença de Gaucher/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Glucosilceramidase/deficiência , Ibuprofeno/uso terapêutico , Imunoglobulina G/metabolismo , Molécula 1 de Adesão Intercelular/metabolismo , Proteínas de Filamentos Intermediários/genética , Imageamento por Ressonância Magnética , Camundongos , Camundongos Transgênicos , Proteínas dos Microfilamentos/metabolismo , Microglia/patologia , Proteínas do Tecido Nervoso/genética , Nestina , Neurônios/efeitos dos fármacos , TATA Box , Tirosina/análogos & derivados , Tirosina/metabolismo , Molécula 1 de Adesão de Célula Vascular/metabolismoRESUMO
Gaucher disease is an inherited metabolic disease caused by the defective activity of the lysosomal enzyme, glucosylceramidase (GlcCerase), which is responsible for the last step in the degradation of complex glycosphingolipids. As a result, glucosylceramide (GlcCer) accumulates intracellularly. Little is known about the mechanisms by which GlcCer accumulation leads to Gaucher disease, particularly for the types of the disease in which severe neuropathology occurs. We now summarize recent advances in this area and in particular focus in the biochemical and cellular pathways that may cause neuronal defects. Most recent work has taken advantage of newly available mouse models, which mimic to a large extent human disease progression. Finally, we discuss observations of a genetic link between Gaucher disease and Parkinson's disease and discuss how this link has stimulated research into the basic biology of the previously underappreciated glycosphingolipid, GlcCer.
Assuntos
Encéfalo/metabolismo , Doença de Gaucher/metabolismo , Neurônios/metabolismo , Animais , Encéfalo/patologia , Modelos Animais de Doenças , Doença de Gaucher/genética , Doença de Gaucher/patologia , Doença de Gaucher/terapia , Predisposição Genética para Doença , Glucosilceramidas/metabolismo , Humanos , Camundongos , Neurônios/patologia , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Transdução de SinaisRESUMO
Virus-induced CNS diseases impose a considerable human health burden worldwide. For many viral CNS infections, neither antiviral drugs nor vaccines are available. In this study, we examined whether the synthesis of glycosphingolipids, major membrane lipid constituents, could be used to establish an antiviral therapeutic target. We found that neuroinvasive Sindbis virus altered the sphingolipid levels early after infection in vitro and increased the levels of gangliosides GA1 and GM1 in the sera of infected mice. The alteration in the sphingolipid levels appears to play a role in neuroinvasive Sindbis virus replication, as treating infected cells with UDP-glucose ceramide glucosyltransferase (UGCG) inhibitors reduced the replication rate. Moreover, the UGCG inhibitor GZ-161 increased the survival rates of Sindbis-infected mice, most likely by reducing the detrimental immune response activated by sphingolipids in the brains of Sindbis virus-infected mice. These findings suggest a role for glycosphingolipids in the host immune response against neuroinvasive Sindbis virus and suggest that UGCG inhibitors should be further examined as antiviral therapeutics for viral infections of the CNS.
RESUMO
The neuronopathic forms of the human inherited metabolic disorder, Gaucher disease (GD), are characterized by severe neuronal loss, astrogliosis and microglial proliferation, but the cellular and molecular pathways causing these changes are not known. Recently, a mouse model of neuronopathic GD was generated in which glucocerebrosidase deficiency is limited to neural and glial progenitor cells. We now show significant changes in the levels and in the distribution of cathepsins in the brain of this mouse model. Cathepsin mRNA expression was significantly elevated by up to approximately 10-fold, with the time-course of the increase correlating with the progression of disease severity. Cathepsin activity and protein levels were also elevated. Significant changes in cathepsin D distribution in the brain were detected, with cathepsin D elevated in areas where neuronal loss, astrogliosis and microgliosis were observed, such as in layer V of the cerebral cortex, the lateral globus pallidus and in various nuclei in the thalamus, brain regions known to be affected in the disease. Cathepsin D elevation was greatest in microglia and also noticeable in astrocytes. The distribution of cathepsin D was altered in neurons in a manner consistent with its release from the lysosome to the cytosol. Remarkably, ibubrofen treatment significantly reduced cathepsin D mRNA levels in the cortex of Gaucher mice. Finally, cathepsin levels were also altered in mouse models of a number of other sphingolipidoses. Our findings suggest the involvement of cathepsins in the neuropathology of neuronal forms of GD and of other lysosomal storage diseases, and are consistent with a crucial role for reactive microglia in neuronal degeneration in these diseases.
Assuntos
Catepsinas/genética , Catepsinas/metabolismo , Doença de Gaucher/metabolismo , Expressão Gênica , Esfingolipidoses/genética , Animais , Encéfalo/metabolismo , Modelos Animais de Doenças , Feminino , Doença de Gaucher/genética , Humanos , Masculino , Camundongos , Camundongos Knockout , Neurônios/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transporte Proteico , Esfingolipidoses/metabolismoRESUMO
Understanding pathways that might impact coronavirus disease 2019 (COVID-19) manifestations and disease outcomes is necessary for better disease management and for therapeutic development. Here, we analyzed alterations in sphingolipid (SL) levels upon infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). SARS-CoV-2 infection induced elevation of SL levels in both cells and sera of infected mice. A significant increase in glycosphingolipid levels was induced early post SARS-CoV-2 infection, which was essential for viral replication. This elevation could be reversed by treatment with glucosylceramide synthase inhibitors. Levels of sphinganine, sphingosine, GA1, and GM3 were significantly increased in both cells and the murine model upon SARS-CoV-2 infection. The potential involvement of SLs in COVID-19 pathology is discussed.
Assuntos
COVID-19/metabolismo , Modelos Animais de Doenças , Esfingolipídeos/metabolismo , Replicação Viral/fisiologia , Animais , COVID-19/prevenção & controle , COVID-19/virologia , Chlorocebus aethiops , Cromatografia Líquida/métodos , Dioxanos/farmacologia , Gangliosídeos/sangue , Gangliosídeos/metabolismo , Glucosiltransferases/antagonistas & inibidores , Glucosiltransferases/metabolismo , Humanos , Espectrometria de Massas/métodos , Camundongos Transgênicos , Pirrolidinas/farmacologia , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/fisiologia , Esfingolipídeos/sangue , Esfingosina/análogos & derivados , Esfingosina/sangue , Esfingosina/metabolismo , Células Vero , Replicação Viral/efeitos dos fármacosRESUMO
Lysosomal storage diseases (LSDs), of which about 50 are known, are caused by the defective activity of lysosomal proteins, resulting in accumulation of unmetabolized substrates. As a result, a variety of pathogenic cascades are activated such as altered calcium homeostasis, oxidative stress, inflammation, altered lipid trafficking, autophagy, endoplasmic reticulum stress, and autoimmune responses. Some of these pathways are common to many LSDs, whereas others are only altered in a subset of LSDs. We now review how these cascades impact upon LSD pathology and suggest how intervention in the pathways may lead to novel therapeutic approaches.
Assuntos
Doenças por Armazenamento dos Lisossomos/genética , Antígenos CD/metabolismo , Doenças Autoimunes/epidemiologia , Autofagia , Cálcio/metabolismo , Retículo Endoplasmático/metabolismo , Radicais Livres/metabolismo , Gangliosídeo G(M2)/metabolismo , Gangliosídeo G(M3)/metabolismo , Doença de Gaucher/metabolismo , Humanos , Lactosilceramidas/metabolismo , Doenças por Armazenamento dos Lisossomos/complicações , Doenças por Armazenamento dos Lisossomos/metabolismo , Doenças por Armazenamento dos Lisossomos/patologia , Lisossomos/metabolismo , Mitocôndrias/metabolismo , Estresse OxidativoRESUMO
Mouse monoclonal antibodies were raised against plague disease biomarkers: the bacterial capsular protein fraction 1 (F1) and the low-calcium response-LcrV virulence factor (Vag). A novel tandem assay, employing BioLayer Interferometry (BLI), enabled the isolation of antibodies against four different epitopes on Vag. The tandem assay was carried out with hybridoma supernatants, circumventing the need for antibody purification. The BioLayer assay was further adopted for characterization of epitope-repetitive antigens, enabling the discovery of two unique epitopes on F1. The selected antibodies were purified and applied as "oligo-clonal" reagents for the immuno-detection of both biomarkers. The developed Homogenous Time Resolved Fluorescence (HTRF) tests were short (10 min) and simple (no washing steps), allowing for detection of 10 ng/mL F1 and 2.5 ng/mL Vag. The tests were successfully applied for detection of disease biomarkers produced by various Y. pestis strains during growth in blood culture vials.
RESUMO
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the ongoing coronavirus disease 2019 (COVID-19) pandemic. The continued spread of SARS-CoV-2 increases the probability of influenza/SARS-CoV-2 coinfection, which may result in severe disease. In this study, we examine the disease outcome of influenza A virus (IAV) and SARS-CoV-2 coinfection in K18-hACE2 mice. Our data indicate enhance susceptibility of IAV-infected mice to developing severe disease upon coinfection with SARS-CoV-2 two days later. In contrast to nonfatal influenza and lower mortality rates due to SARS-CoV-2 alone, this coinfection results in severe morbidity and nearly complete mortality. Coinfection is associated with elevated influenza viral loads in respiratory organs. Remarkably, prior immunity to influenza, but not to SARS-CoV-2, prevents severe disease and mortality. This protection is antibody-dependent. These data experimentally support the necessity of seasonal influenza vaccination for reducing the risk of severe influenza/COVID-19 comorbidity during the COVID-19 pandemic.
Assuntos
COVID-19/imunologia , COVID-19/virologia , Coinfecção/imunologia , Coinfecção/virologia , Imunidade , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/virologia , SARS-CoV-2/imunologia , Enzima de Conversão de Angiotensina 2/metabolismo , Animais , Anticorpos Antivirais/imunologia , COVID-19/patologia , Linhagem Celular , Modelos Animais de Doenças , Feminino , Humanos , Inflamação/genética , Pulmão/patologia , Pulmão/virologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Regulação para Cima/genética , Carga Viral/imunologiaRESUMO
The coronavirus disease 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), exhibits high levels of mortality and morbidity and has dramatic consequences on human life, sociality and global economy. Neutralizing antibodies constitute a highly promising approach for treating and preventing infection by this novel pathogen. In the present study, we characterize and further evaluate the recently identified human monoclonal MD65 antibody for its ability to provide protection against a lethal SARS-CoV-2 infection of K18-hACE2 transgenic mice. Eighty percent of the untreated mice succumbed 6-9 days post-infection, while administration of the MD65 antibody as late as 3 days after exposure rescued all infected animals. In addition, the efficiency of the treatment is supported by prevention of morbidity and ablation of the load of infective virions in the lungs of treated animals. The data demonstrate the therapeutic value of human monoclonal antibodies as a life-saving treatment for severe COVID-19 infection.