RESUMO
Insectivorous Old World horseshoe bats (Rhinolophus spp.) are the likely source of the ancestral SARS-CoV-2 prior to its spillover into humans and causing the COVID-19 pandemic. Natural coronavirus infections of bats appear to be principally confined to the intestines, suggesting fecal-oral transmission; however, little is known about the biology of SARS-related coronaviruses in bats. Previous experimental challenges of Egyptian fruit bats (Rousettus aegyptiacus) resulted in limited infection restricted to the respiratory tract, whereas insectivorous North American big brown bats (Eptesicus fuscus) showed no evidence of infection. In the present study, we challenged Jamaican fruit bats (Artibeus jamaicensis) with SARS-CoV-2 to determine their susceptibility. Infection was confined to the intestine for only a few days with prominent viral nucleocapsid antigen in epithelial cells, and mononuclear cells of the lamina propria and Peyer's patches, but with no evidence of infection of other tissues; none of the bats showed visible signs of disease or seroconverted. Expression levels of ACE2 were low in the lungs, which may account for the lack of pulmonary infection. Bats were then intranasally inoculated with a replication-defective adenovirus encoding human ACE2 and 5 days later challenged with SARS-CoV-2. Viral antigen was prominent in lungs for up to 14 days, with loss of pulmonary cellularity during this time; however, the bats did not exhibit weight loss or visible signs of disease. From day 7, bats had low to moderate IgG antibody titers to spike protein by ELISA, and one bat on day 10 had low-titer neutralizing antibodies. CD4+ helper T cells became activated upon ex vivo recall stimulation with SARS-CoV-2 nucleocapsid peptide library and exhibited elevated mRNA expression of the regulatory T cell cytokines interleukin-10 and transforming growth factor-ß, which may have limited inflammatory pathology. Collectively, these data show that Jamaican fruit bats are poorly susceptible to SARS-CoV-2 but that expression of human ACE2 in their lungs leads to robust infection and an adaptive immune response with low-titer antibodies and a regulatory T cell-like response that may explain the lack of prominent inflammation in the lungs. This model will allow for insight of how SARS-CoV-2 infects bats and how bat innate and adaptive immune responses engage the virus without overt clinical disease.
Assuntos
COVID-19 , Quirópteros , Animais , Humanos , SARS-CoV-2 , Enzima de Conversão de Angiotensina 2 , Pandemias , Jamaica , Linfócitos T ReguladoresRESUMO
Parkinson's disease (PD) is the most common neurodegenerative movement disorder worldwide, with a greater prevalence in men than women. The etiology of PD is largely unknown, although environmental exposures and neuroinflammation are linked to protein misfolding and disease progression. Activated microglia are known to promote neuroinflammation in PD, but how environmental agents interact with specific innate immune signaling pathways in microglia to stimulate conversion to a neurotoxic phenotype is not well understood. To determine how nuclear factor kappa B (NF-κB) signaling dynamics in microglia modulate neuroinflammation and dopaminergic neurodegeneration, we generated mice deficient in NF-κB activation in microglia (CX3CR1-Cre::IKK2fl/fl ) and exposed them to 2.5 mg/kg/day of rotenone for 14 days, followed by a 14-day post-lesioning incubation period. We postulated that inhibition of NF-κB signaling in microglia would reduce overall inflammatory injury in lesioned mice. Subsequent analysis indicated decreased expression of the NF-κB-regulated autophagy gene, sequestosome 1 (p62), in microglia, which is required for targeting ubiquitinated α-synuclein (α-syn) for lysosomal degradation. Knock-out animals had increased accumulation of misfolded α-syn within microglia, despite an overall reduction in neurodegeneration. Interestingly, this occurred more prominently in males. These data suggest that microglia play key biological roles in the degradation and clearance of misfolded α-syn and this process works in concert with the innate immune response associated with neuroinflammation. Importantly, the accumulation of misfolded α-syn protein aggregates alone did not increase neurodegeneration following exposure to rotenone but required the NF-κB-dependent inflammatory response in microglia.
Assuntos
Doenças Neurodegenerativas , Doença de Parkinson , Masculino , Feminino , Camundongos , Animais , Doença de Parkinson/genética , alfa-Sinucleína/metabolismo , NF-kappa B/metabolismo , Rotenona/toxicidade , Rotenona/metabolismo , Microglia/metabolismo , Doenças Neuroinflamatórias , Doenças Neurodegenerativas/metabolismo , Autofagia , Neurônios Dopaminérgicos/metabolismoRESUMO
Coronavirus disease-19 (COVID-19) emerged in late 2019 in China and rapidly became pandemic. As with other coronaviruses, a preponderance of evidence suggests the virus originated in horseshoe bats (Rhinolophus spp.) and may have infected an intermediate host prior to spillover into humans. A significant concern is that SARS-CoV-2 could become established in secondary reservoir hosts outside of Asia. To assess this potential, we challenged deer mice (Peromyscus maniculatus) with SARS-CoV-2 and found robust virus replication in the upper respiratory tract, lungs and intestines, with detectable viral RNA for up to 21 days in oral swabs and 6 days in lungs. Virus entry into the brain also occurred, likely via gustatory-olfactory-trigeminal pathway with eventual compromise to the blood-brain barrier. Despite this, no conspicuous signs of disease were observed, and no deer mice succumbed to infection. Expression of several innate immune response genes were elevated in the lungs, including IFNα, IFNß, Cxcl10, Oas2, Tbk1 and Pycard. Elevated CD4 and CD8ß expression in the lungs was concomitant with Tbx21, IFNγ and IL-21 expression, suggesting a type I inflammatory immune response. Contact transmission occurred from infected to naive deer mice through two passages, showing sustained natural transmission and localization into the olfactory bulb, recapitulating human neuropathology. In the second deer mouse passage, an insertion of 4 amino acids occurred to fixation in the N-terminal domain of the spike protein that is predicted to form a solvent-accessible loop. Subsequent examination of the source virus from BEI Resources determined the mutation was present at very low levels, demonstrating potent purifying selection for the insert during in vivo passage. Collectively, this work has determined that deer mice are a suitable animal model for the study of SARS-CoV-2 respiratory disease and neuropathogenesis, and that they have the potential to serve as secondary reservoir hosts in North America.
Assuntos
COVID-19/fisiopatologia , COVID-19/transmissão , Peromyscus/virologia , Doenças dos Roedores/transmissão , Animais , Encéfalo/patologia , Encéfalo/virologia , COVID-19/patologia , Modelos Animais de Doenças , Reservatórios de Doenças , Suscetibilidade a Doenças , Feminino , Masculino , Doenças dos Roedores/patologia , Doenças dos Roedores/virologia , Glicoproteína da Espícula de Coronavírus/genética , Replicação ViralRESUMO
Rotenone is a naturally occurring insecticide that inhibits mitochondrial complex I and leads to neurochemical and neuropathological deficits closely resembling those in Parkinson's disease (PD). Deficits include loss of dopaminergic neurons (DAn) in the substantia nigra pars compacta (SNpc), decreased dopamine levels and aggregation of misfolded alpha-synuclein (p129). In rat models of rotenone-induced parkinsonism, the progression of neuronal injury has been associated with activation of microglia and astrocytes. However, these neuroinflammatory changes have been challenging to study in mice, in part because the systemic rotenone exposure model utilized in rats is more toxic to mice. To establish a reproducible murine model of rotenone-induced PD, we therefore investigated the progression of neuroinflammation, protein aggregation and DAn loss in C57Bl/6 mice by exposing animals to 2.5 mg/kg/day rotenone for 14 days, followed by a two-week period where neuroinflammation is allowed to progress. Our results indicate that initial cellular dysfunction leads to increased formation of proteinase K-resistant p129 aggregates in the caudate-putamen and SNpc. Clearance of these aggregates was region- and cell type-specific, with the early appearance of reactive astrocytes coinciding with accumulation of p129 in the SNpc. Phagocytic microglial cells containing p129 aggregates were observed proximal to p129+ DAn in the SNpc. The majority of neuronal loss in the SNpc occurred during the two-week period after rotenone exposure, subsequent to the peak of microglia and astrocyte activation, as well as the peak of p129 aggregation. A secondary peak of p129 coincided with neurodegeneration at later timepoints. These data indicate that systemic exposure to rotenone in C57Bl/6 mice causes progressive accumulation and regional spread of p129 aggregates that precede maximal loss of DAn. Thus, activation of glial cells and aggregation of p129 appear to drive neuronal loss following neurotoxic stress imposed by exposure to rotenone.
Assuntos
Neurônios Dopaminérgicos , Rotenona , Animais , Neurônios Dopaminérgicos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Microglia/metabolismo , Agregados Proteicos , Ratos , Rotenona/toxicidade , Substância Negra/metabolismo , alfa-Sinucleína/metabolismoRESUMO
The modified phytochemical derivative, 1,1-bis(3'-indolyl)-1-(p-chlorophenyl) methane (C-DIM12), has been identified as a potential therapeutic platform based on its capacity to improve disease outcomes in models of neurodegeneration and cancer. However, comprehensive safety studies investigating pathology and off-target binding have not been conducted. To address this, we administered C-DIM12 orogastrically to outbred male CD-1 mice for 7 days (50 mg/kg/day, 200 mg/kg/day, and 300 mg/kg/day) and investigated changes in hematology, clinical chemistry, and whole-body tissue pathology. We also delivered a single dose of C-DIM12 (1 mg/kg, 5 mg/kg, 25 mg/kg, 100 mg/kg, 300 mg/kg, 1,000 mg/kg) orogastrically to male and female beagle dogs and investigated hematology and clinical chemistry, as well as plasma pharmacokinetics over 48-h. Consecutive in-vitro off-target binding through inhibition was performed with 10 µM C-DIM12 against 68 targets in tandem with predictive off-target structural binding capacity. These data show that the highest dose C-DIM12 administered in each species caused modest liver pathology in mouse and dog, whereas lower doses were unremarkable. Off-target screening and predictive modeling of C-DIM12 show inhibition of serine/threonine kinases, calcium signaling, G-protein coupled receptors, extracellular matrix degradation, and vascular and transcriptional regulation pathways. Collectively, these data demonstrate that low doses of C-DIM12 do not induce pathology and are capable of modulating targets relevant to neurodegeneration and cancer.
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Parkinson's disease (PD) is the most common neurodegenerative movement disorder worldwide. Current treatments for PD largely center around dopamine replacement therapies and fail to prevent the progression of pathology, underscoring the need for neuroprotective interventions. Approaches that target neuroinflammation, which occurs prior to dopaminergic neuron (DAn) loss in the substantia nigra (SN), represent a promising therapeutic strategy. The glucocorticoid receptor (GR) has been implicated in the neuropathology of PD and modulates numerous neuroinflammatory signaling pathways in the brain. Therefore, we investigated the neuroprotective effects of the novel GR modulator, PT150, in the rotenone mouse model of PD, postulating that inhibition of glial inflammation would protect DAn and reduce accumulation of neurotoxic misfolded âº-synuclein protein. C57Bl/6 mice were exposed to 2.5 mg/kg/day rotenone by intraperitoneal injection for 14 days, immediately followed by oral treatment with 30 mg/kg/day or 100 mg/kg/day PT150 in the 14-day post-lesioning incubation period, during which the majority of DAn loss and α-synuclein (α-syn) accumulation occurs. Our results indicate that treatment with PT150 reduced both loss of DAn and microgliosis in the nigrostriatal pathway. Although morphologic features of astrogliosis were not attenuated, PT150 treatment promoted potentially neuroprotective activity in these cells, including increased phagocytosis of hyperphosphorylated α-syn. Ultimately, PT150 treatment reduced the loss of DAn cell bodies in the SN, but not the striatum, and prohibited intra-neuronal accumulation of α-syn. Together, these data indicate that PT150 effectively reduced SN pathology in the rotenone mouse model of PD.
RESUMO
Parkinson's disease (PD) is the most common neurodegenerative movement disorder worldwide. Current treatments for PD largely center around dopamine replacement therapies and fail to prevent the progression of pathology, underscoring the need for neuroprotective interventions. Approaches that target neuroinflammation, which occurs prior to dopaminergic neuron (DAn) loss in the substantia nigra (SN), represent a promising therapeutic strategy. The glucocorticoid receptor (GR) has been implicated in the neuropathology of PD and modulates numerous neuroinflammatory signaling pathways in the brain. Therefore, we investigated the neuroprotective effects of the novel GR modulator, PT150, in the rotenone mouse model of PD, postulating that inhibition of glial inflammation would protect DAn and reduce accumulation of neurotoxic misfolded âº-synuclein protein. C57Bl/6 mice were exposed to 2.5â¯mg/kg/day rotenone by intraperitoneal injection for 14 days. Upon completion of rotenone dosing, mice were orally treated at day 15 with 30â¯mg/kg/day or 100â¯mg/kg/day PT150 in the 14-day post-lesioning incubation period, during which the majority of DAn loss and α-synuclein (α-syn) accumulation occurs. Our results indicate that treatment with PT150 reduced both loss of DAn and microgliosis in the nigrostriatal pathway. Although morphologic features of astrogliosis were not attenuated, PT150 treatment promoted potentially neuroprotective activity in these cells, including increased phagocytosis of hyperphosphorylated α-syn. Ultimately, PT150 treatment reduced the loss of DAn cell bodies in the SN, but not the striatum, and prohibited intra-neuronal accumulation of α-syn. Together, these data indicate that PT150 effectively reduced SN pathology in the rotenone mouse model of PD.
Assuntos
Neurônios Dopaminérgicos , Camundongos Endogâmicos C57BL , Fármacos Neuroprotetores , Receptores de Glucocorticoides , Rotenona , alfa-Sinucleína , Animais , Rotenona/toxicidade , Fármacos Neuroprotetores/farmacologia , Camundongos , Masculino , Neurônios Dopaminérgicos/efeitos dos fármacos , Neurônios Dopaminérgicos/patologia , Neurônios Dopaminérgicos/metabolismo , Receptores de Glucocorticoides/metabolismo , alfa-Sinucleína/metabolismo , Substância Negra/efeitos dos fármacos , Substância Negra/patologia , Substância Negra/metabolismo , Transtornos Parkinsonianos/tratamento farmacológico , Transtornos Parkinsonianos/induzido quimicamente , Transtornos Parkinsonianos/metabolismo , Transtornos Parkinsonianos/patologia , Modelos Animais de Doenças , FenantrenosRESUMO
Bats are natural reservoir hosts of many important zoonotic viruses but because there are few immunological reagents and breeding colonies available for infectious disease research, little is known about their immune responses to infection. We established a breeding colony Jamaican fruit bats ( Artibeus jamaicensis ) to study bat virology and immunology. The species is used as a natural reservoir model for H18N11 influenza A virus, and as a surrogate model for SARS-CoV-2, MERS-CoV and Tacaribe virus. As part of our ongoing efforts to develop this model organism, we sought to identify commercially available monoclonal antibodies (mAb) for profiling Jamaican fruit bat lymphocytes. We identified several cross-reactive mAb that can be used to identify T and B cells; however, we were unable to identify mAb for three informative T cell markers, CD3γ, CD4 and CD8α. We targeted these markers for the generation of hybridomas, and identified several clones to each that can be used with flow cytometry and fluorescence microscopy. Specificity of the monoclonal antibodies was validated by sorting lymphocytes, followed by PCR identification of confirmatory transcripts. Spleens of Jamaican fruit bats possess about half the number of T cells than do human or mouse spleens, and we identified an unusual population of cells that expressed the B cell marker CD19 and the T cell marker CD3. The availability of these monoclonal antibodies will permit a more thorough examination of adaptive immune responses in Jamaican fruit bats that should help clarify how the bats control viral infections and without disease. Importance: Bats naturally host a number of viruses without disease, but which can cause significant disease in humans. Virtually nothing is known about adaptive immune responses in bats because of a lack of immunological tools to examine such responses. We have begun to address this deficiency by identifying several commercially available monoclonal antibodies to human and mouse antigens that are cross-reactive to Jamaican fruit bat lymphocyte orthologs. We also generated monoclonal antibodies to Jamaican fruit bat CD3γ, CD4 and CD8α that are suitable for identifying T cell subsets by flow cytometry and immunofluorescent staining of fixed tissues. Together, these reagents will allow a more detailed examination of lymphocyte populations in Jamaican fruit bats.
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A 6-year-old female spayed Great Dane was evaluated for acute onset cluster seizures. Magnetic resonance imaging (MRI) identified a mass in the olfactory bulbs with a large mucoid component caudal to the primary mass. The mass was removed via transfrontal craniotomy and histopathology revealed a tyrosine crystalline-rich, fibrous meningioma with a high mitotic index. Repeat MRI at 6 months showed no detectable tumor regrowth. The dog is clinically normal with no seizures at the time of publication 10 months after surgery. This meningioma subtype is rare in humans. This unique meningioma occurred in a dog of younger age and uncommon breed for intracranial meningioma. Biological progression of this tumor subtype is unknown; however, growth rate might be slow despite the high mitotic index.
Assuntos
Doenças do Cão , Neoplasias Meníngeas , Meningioma , Humanos , Feminino , Animais , Cães , Meningioma/diagnóstico por imagem , Meningioma/cirurgia , Meningioma/veterinária , Neoplasias Meníngeas/diagnóstico por imagem , Neoplasias Meníngeas/cirurgia , Neoplasias Meníngeas/veterinária , Convulsões/veterinária , Craniotomia/métodos , Craniotomia/veterinária , Imageamento por Ressonância Magnética/veterinária , Tirosina , Doenças do Cão/diagnóstico por imagem , Doenças do Cão/cirurgiaRESUMO
Insectivorous Old World horseshoe bats ( Rhinolophus spp.) are the likely source of the ancestral SARS-CoV-2 prior to its spillover into humans and causing the COVID-19 pandemic. Natural coronavirus infections of bats appear to be principally confined to the intestines, suggesting fecal-oral transmission; however, little is known about the biology of SARS-related coronaviruses in bats. Previous experimental challenges of Egyptian fruit bats ( Rousettus aegyptiacus ) resulted in limited infection restricted to the respiratory tract, whereas insectivorous North American big brown bats ( Eptesicus fuscus ) showed no evidence of infection. In the present study, we challenged Jamaican fruit bats ( Artibeus jamaicensis ) with SARS-CoV-2 to determine their susceptibility. Infection was confined to the intestine for only a few days with prominent viral nucleocapsid antigen in epithelial cells, and mononuclear cells of the lamina propria and Peyer's patches, but with no evidence of infection of other tissues; none of the bats showed visible signs of disease or seroconverted. Expression levels of ACE2 were low in the lungs, which may account for the lack of pulmonary infection. Bats were then intranasally inoculated with a replication-defective adenovirus encoding human ACE2 and 5 days later challenged with SARS-CoV-2. Viral antigen was prominent in lungs for up to 14 days, with loss of pulmonary cellularity during this time; however, the bats did not exhibit weight loss or visible signs of disease. From day 7, bats had low to moderate IgG antibody titers to spike protein by ELISA, and one bat on day 10 had low-titer neutralizing antibodies. CD4 + helper T cells became activated upon ex vivo recall stimulation with SARS-CoV-2 nucleocapsid peptide library and exhibited elevated mRNA expression of the regulatory T cell cytokines interleukin-10 and transforming growth factor-ß, which may have limited inflammatory pathology. Collectively, these data show that Jamaican fruit bats are poorly susceptibility to SARS-CoV-2 but that expression of human ACE2 in their lungs leads to robust infection and an adaptive immune response with low-titer antibodies and a regulatory T cell-like response that may explain the lack of prominent inflammation in the lungs. This model will allow for insight of how SARS-CoV-2 infects bats and how bat innate and adaptive immune responses engage the virus without overt clinical disease. Author Summary: Bats are reservoir hosts of many viruses that infect humans, yet little is known about how they host these viruses, principally because of a lack of relevant and susceptible bat experimental infection models. Although SARS-CoV-2 originated in bats, no robust infection models of bats have been established. We determined that Jamaican fruit bats are poorly susceptible to SARS-CoV-2; however, their lungs can be transduced with human ACE2, which renders them susceptible to SARS-CoV-2. Despite robust infection of the lungs and diminishment of pulmonary cellularity, the bats showed no overt signs of disease and cleared the infection after two weeks. Despite clearance of infection, only low-titer antibody responses occurred and only a single bat made neutralizing antibody. Assessment of the CD4 + helper T cell response showed that activated cells expressed the regulatory T cell cytokines IL-10 and TGFß that may have tempered pulmonary inflammation.
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Despite significant research efforts, treatment options for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remain limited. This is due in part to a lack of therapeutics that increase host defense to the virus. Replication of SARS-CoV-2 in lung tissue is associated with marked infiltration of macrophages and activation of innate immune inflammatory responses that amplify tissue injury. Antagonists of the androgen (AR) and glucocorticoid (GR) receptors have shown efficacy in models of COVID-19 and in clinical studies because the cell surface proteins required for viral entry, angiotensin converting enzyme 2 (ACE2) and the transmembrane protease, serine 2 (TMPRSS2), are transcriptionally regulated by these receptors. We postulated that the GR and AR modulator, PT150, would reduce infectivity of SARS-CoV-2 and prevent inflammatory lung injury in the Syrian golden hamster model of COVID-19 by down-regulating expression of critical genes regulated through these receptors. Animals were infected intranasally with 2.5 × 104 TCID50/ml equivalents of SARS-CoV-2 (strain 2019-nCoV/USA-WA1/2020) and PT150 was administered by oral gavage at 30 and 100 mg/Kg/day for a total of 7 days. Animals were examined at 3, 5 and 7 days post-infection (DPI) for lung histopathology, viral load and production of proteins regulating the progression of SARS-CoV-2 infection. Results indicated that oral administration of PT150 caused a dose-dependent decrease in replication of SARS-CoV-2 in lung, as well as in expression of ACE2 and TMPRSS2. Lung hypercellularity and infiltration of macrophages and CD4+ T-cells were dramatically decreased in PT150-treated animals, as was tissue damage and expression of IL-6. Molecular docking studies suggest that PT150 binds to the co-activator interface of the ligand-binding domain of both AR and GR, thereby acting as an allosteric modulator and transcriptional repressor of these receptors. Phylogenetic analysis of AR and GR revealed a high degree of sequence identity maintained across multiple species, including humans, suggesting that the mechanism of action and therapeutic efficacy observed in Syrian hamsters would likely be predictive of positive outcomes in patients. PT150 is therefore a strong candidate for further clinical development for the treatment of COVID-19 across variants of SARS-CoV-2.
Assuntos
Antivirais/farmacologia , Tratamento Farmacológico da COVID-19 , Glucocorticoides/metabolismo , Imunidade Inata/efeitos dos fármacos , Inflamação/tratamento farmacológico , Receptores Androgênicos/metabolismo , Internalização do Vírus/efeitos dos fármacos , Animais , COVID-19/metabolismo , Modelos Animais de Doenças , Feminino , Inflamação/metabolismo , Inflamação/virologia , Pulmão/virologia , Masculino , Mesocricetus , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/metabolismo , Serina Endopeptidases/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Carga Viral/efeitos dos fármacosRESUMO
Viral infection of the central nervous system (CNS) can cause lasting neurological decline in surviving patients and can present with symptoms resembling Parkinson's disease (PD). The mechanisms underlying postencephalitic parkinsonism remain unclear but are thought to involve increased innate inflammatory signaling in glial cells, resulting in persistent neuroinflammation. We therefore studied the role of glial cells in regulating neuropathology in postencephalitic parkinsonism by studying the involvement of astrocytes in loss of dopaminergic neurons and aggregation of α-synuclein protein following infection with western equine encephalitis virus (WEEV). Infections were conducted in both wildtype mice and in transgenic mice lacking NFκB inflammatory signaling in astrocytes. For 2 months following WEEV infection, we analyzed glial activation, neuronal loss and protein aggregation across multiple brain regions, including the substantia nigra pars compacta (SNpc). These data revealed that WEEV induces loss of SNpc dopaminergic neurons, persistent activation of microglia and astrocytes that precipitates widespread aggregation of α-synuclein in the brain of C57BL/6 mice. Microgliosis and macrophage infiltration occurred prior to activation of astrocytes and was followed by opsonization of âº-synuclein protein aggregates in the cortex, hippocampus and midbrain by the complement protein, C3. Astrocyte-specific NFκB knockout mice had reduced gliosis, α-synuclein aggregate formation and neuronal loss. These data suggest that astrocytes play a critical role in initiating PD-like pathology following encephalitic infection with WEEV through innate immune inflammatory pathways that damage dopaminergic neurons, possibly by hindering clearance of âº-synuclein aggregates. Inhibiting glial inflammatory responses could therefore represent a potential therapy strategy for viral parkinsonism.
Assuntos
Astrócitos/metabolismo , Neurônios Dopaminérgicos/metabolismo , Encefalite Viral/metabolismo , Mediadores da Inflamação/metabolismo , Agregados Proteicos/fisiologia , alfa-Sinucleína/metabolismo , Animais , Astrócitos/imunologia , Neurônios Dopaminérgicos/imunologia , Vírus da Encefalite Equina do Oeste/imunologia , Vírus da Encefalite Equina do Oeste/metabolismo , Encefalite Viral/imunologia , Feminino , Humanos , Mediadores da Inflamação/imunologia , Masculino , Camundongos , Camundongos Knockout , Transdução de Sinais/fisiologiaRESUMO
The emergence of COVID-19 has led to a pandemic that has caused millions of cases of disease, variable morbidity and hundreds of thousands of deaths. Currently, only remdesivir and dexamethasone have demonstrated limited efficacy, only slightly reducing disease burden, thus novel approaches for clinical management of COVID-19 are needed. We identified a panel of human monoclonal antibody clones from a yeast display library with specificity to the SARS-CoV-2 spike protein receptor binding domain that neutralized the virus in vitro. Administration of the lead antibody clone to Syrian hamsters challenged with SARS-CoV-2 significantly reduced viral load and histopathology score in the lungs. Moreover, the antibody interrupted monocyte infiltration into the lungs, which may have contributed to the reduction of disease severity by limiting immunopathological exacerbation. The use of this antibody could provide an important therapy for treatment of COVID-19 patients.
Assuntos
Anticorpos Monoclonais , Anticorpos Neutralizantes , Anticorpos Antivirais , Tratamento Farmacológico da COVID-19 , COVID-19 , Imunoglobulina G , SARS-CoV-2/imunologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/farmacologia , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/farmacologia , COVID-19/sangue , COVID-19/imunologia , Chlorocebus aethiops , Humanos , Imunoglobulina G/imunologia , Imunoglobulina G/farmacologia , Masculino , Mesocricetus , Índice de Gravidade de Doença , Células Vero , Carga Viral/efeitos dos fármacos , Carga Viral/imunologiaRESUMO
The emergence of COVID-19 has led to a pandemic that has caused millions of cases of disease, variable morbidity and hundreds of thousands of deaths. Currently, only remdesivir and dexamethasone have demonstrated limited efficacy, only slightly reducing disease burden, thus novel approaches for clinical management of COVID-19 are needed. We identified a panel of human monoclonal antibody clones from a yeast display library with specificity to the SARS-CoV-2 spike protein receptor binding domain that neutralized the virus in vitro . Administration of the lead antibody clone to Syrian hamsters challenged with SARS-CoV-2 significantly reduced viral load and histopathology score in the lungs. Moreover, the antibody interrupted monocyte infiltration into the lungs, which may have contributed to the reduction of disease severity by limiting immunopathological exacerbation. The use of this antibody could provide an important therapy for treatment of COVID-19 patients.
RESUMO
Coronavirus disease-19 (COVID-19) emerged in November, 2019 in China and rapidly became pandemic. As with other coronaviruses, a preponderance of evidence suggests the virus originated in horseshoe bats (Rhinolophus spp.) and likely underwent a recombination event in an intermediate host prior to entry into human populations. A significant concern is that SARS-CoV-2 could become established in secondary reservoir hosts outside of Asia. To assess this potential, we challenged deer mice (Peromyscus maniculatus) with SARS-CoV-2 and found robust virus replication in the upper respiratory tract, lungs and intestines, with detectable viral RNA for up to 21 days in oral swabs and 14 days in lungs. Virus entry into the brain also occurred, likely via gustatory-olfactory-trigeminal pathway with eventual compromise to the blood brain barrier. Despite this, no conspicuous signs of disease were observed and no deer mice succumbed to infection. Expression of several innate immune response genes were elevated in the lungs, notably IFNα, Cxcl10, Oas2, Tbk1 and Pycard. Elevated CD4 and CD8ß expression in the lungs was concomitant with Tbx21, IFNγ and IL-21 expression, suggesting a type I inflammatory immune response. Contact transmission occurred from infected to naive deer mice through two passages, showing sustained natural transmission. In the second deer mouse passage, an insertion of 4 amino acids occurred to fixation in the N-terminal domain of the spike protein that is predicted to form a solvent-accessible loop. Subsequent examination of the source virus from BEI Resources indicated the mutation was present at very low levels, demonstrating potent purifying selection for the insert during in vivo passage. Collectively, this work has determined that deer mice are a suitable animal model for the study of SARS-CoV-2 pathogenesis, and that they have the potential to serve as secondary reservoir hosts that could lead to periodic outbreaks of COVID-19 in North America.
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Neuroinvasive infections with mosquito-borne alphaviruses such as Western equine encephalitis virus (WEEV) can cause post-encephalitic parkinsonism. To understand the mechanisms underlying these neurological effects, we examined the capacity of WEEV to induce progressive neurodegeneration in outbred CD-1 mice following non-lethal encephalitic infection. Animals were experientally infected with recombinant WEEV expressing firefly luciferase or dsRed (RFP) reporters and the extent of viral replication was controlled using passive immunotherapy. WEEV spread along the neuronal axis from the olfactory bulb to the entorhinal cortex, hippocampus and basal midbrain by 4 days post infection (DPI). Infection caused activation of microglia and astrocytes, selective loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) and neurobehavioral abnormalities. After 8 weeks, surviving mice displayed continued loss of dopamine neurons in the SNpc, lingering glial cell activation and gene expression profiles consistent with a neurodegenerative phenotype. Strikingly, prominent proteinase K-resistant protein aggregates were present in the the entorhinal cortex, hippocampus and basal midbrain that stained positively for phospho-serine129 α-synuclein (SNCA). These results indicate that WEEV may cause lasting neurological deficits through a severe neuroinflammatory response promoting both neuronal injury and protein aggregation in surviving individuals.