RESUMO
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a pandemic with millions of human infections. One limitation to the evaluation of potential therapies and vaccines to inhibit SARS-CoV-2 infection and ameliorate disease is the lack of susceptible small animals in large numbers. Commercially available laboratory strains of mice are not readily infected by SARS-CoV-2 because of species-specific differences in their angiotensin-converting enzyme 2 (ACE2) receptors. Here, we transduced replication-defective adenoviruses encoding human ACE2 via intranasal administration into BALB/c mice and established receptor expression in lung tissues. hACE2-transduced mice were productively infected with SARS-CoV-2, and this resulted in high viral titers in the lung, lung pathology, and weight loss. Passive transfer of a neutralizing monoclonal antibody reduced viral burden in the lung and mitigated inflammation and weight loss. The development of an accessible mouse model of SARS-CoV-2 infection and pathogenesis will expedite the testing and deployment of therapeutics and vaccines.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Anticorpos Neutralizantes/uso terapêutico , Anticorpos Antivirais/uso terapêutico , Betacoronavirus/imunologia , Infecções por Coronavirus/terapia , Modelos Animais de Doenças , Pneumonia Viral/terapia , Enzima de Conversão de Angiotensina 2 , Animais , COVID-19 , Chlorocebus aethiops , Infecções por Coronavirus/virologia , Feminino , Células HEK293 , Humanos , Imunização Passiva/métodos , Pulmão/metabolismo , Pulmão/virologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Knockout , Pandemias , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/virologia , SARS-CoV-2 , Transdução Genética , Células Vero , Carga Viral/imunologiaRESUMO
Inherited arrhythmia syndromes (IASs) can cause life-threatening arrhythmias and are responsible for a significant proportion of sudden cardiac deaths (SCDs). Despite progress in the development of devices to prevent SCDs, the precise molecular mechanisms that induce detrimental arrhythmias remain to be fully investigated, and more effective therapies are desirable. In the present study, we screened a large-scale randomly mutagenized mouse library by electrocardiography to establish a disease model of IASs and consequently found one pedigree that exhibited spontaneous ventricular arrhythmias (VAs) followed by SCD within 1 y after birth. Genetic analysis successfully revealed a missense mutation (p.I4093V) of the ryanodine receptor 2 gene to be a cause of the arrhythmia. We found an age-related increase in arrhythmia frequency accompanied by cardiomegaly and decreased ventricular contractility in the Ryr2I4093V/+ mice. Ca2+ signaling analysis and a ryanodine binding assay indicated that the mutant ryanodine receptor 2 had a gain-of-function phenotype and enhanced Ca2+ sensitivity. Using this model, we detected the significant suppression of VA following flecainide or dantrolene treatment. Collectively, we established an inherited life-threatening arrhythmia mouse model from an electrocardiogram-based screen of randomly mutagenized mice. The present IAS model may prove feasible for use in investigating the mechanisms of SCD and assessing therapies.
Assuntos
Taquicardia Ventricular , Camundongos , Animais , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Arritmias Cardíacas/genética , Flecainida , Mutação de Sentido Incorreto , Morte Súbita Cardíaca , MutaçãoRESUMO
Laboratory models are central to microbiology research, advancing the understanding of bacterial physiology by mimicking natural environments, from soil to the human microbiome. When studying host-bacteria interactions, animal models enable investigators to examine bacterial dynamics associated with a host, and in the case of human infections, animal models are necessary to translate basic research into clinical treatments. Efforts toward improving animal infection models are typically based on reproducing host genotypes/phenotypes and disease manifestations, leaving a gap in how well the physiology of microbes reflects their behavior in a human host. Understanding bacterial physiology is vital because it dictates host response and bacterial interactions with antimicrobials. Thus, our goal was to develop an animal model that accurately recapitulates bacterial physiology in human infection. The system we chose to model was a chronic Pseudomonas aeruginosa respiratory infection in cystic fibrosis (CF). To accomplish this goal, we leveraged a framework that we recently developed to evaluate model accuracy by calculating the percentage of bacterial genes that are expressed similarly in a model to how they are expressed in their infection environment. We combined two complementary models of P. aeruginosa infection-an in vitro synthetic CF sputum model (SCFM2) and a mouse acute pneumonia model. This combined model captured the chronic physiology of P. aeruginosa in CF better than the standard mouse infection model, showing the power of a data-driven approach to refining animal models. In addition, the results of this work challenge the assumption that a chronic infection model requires long-term colonization.
Assuntos
Fibrose Cística , Modelos Animais de Doenças , Infecções por Pseudomonas , Pseudomonas aeruginosa , Fibrose Cística/microbiologia , Fibrose Cística/complicações , Pseudomonas aeruginosa/fisiologia , Pseudomonas aeruginosa/patogenicidade , Animais , Infecções por Pseudomonas/microbiologia , Camundongos , Humanos , Infecções Respiratórias/microbiologia , Interações Hospedeiro-Patógeno , Escarro/microbiologiaRESUMO
This review aimed to capture the key findings that animal models have provided around the role of the alternative pathway and amplification loop (AP/AL) in disease. Animal models, particularly mouse models, have been incredibly useful to define the role of complement and the alternative pathway in health and disease; for instance, the use of cobra venom factor and depletion of C3 provided the initial insight that complement was essential to generate an appropriate adaptive immune response. The development of knockout mice have further underlined the importance of the AP/AL in disease, with the FH knockout mouse paving the way for the first anti-complement drugs. The impact from the development of FB, properdin, and C3 knockout mice closely follows this in terms of mechanistic understanding in disease. Indeed, our current understanding that complement plays a role in most conditions at one level or another is rooted in many of these in vivo studies. That C3, in particular, has roles beyond the obvious in innate and adaptive immunity, normal physiology, and cellular functions, with or without other recognized AP components, we would argue, only extends the reach of this arm of the complement system. Humanized mouse models also continue to play their part. Here, we argue that the animal models developed over the last few decades have truly helped define the role of the AP/AL in disease.
Assuntos
Via Alternativa do Complemento , Properdina , Animais , Camundongos , Humanos , Properdina/metabolismo , Imunidade Adaptativa , Camundongos Knockout , Modelos Animais de DoençasRESUMO
Wilson disease (WD) is caused by mutations in the ATP7B gene that encodes a copper (Cu) transporting ATPase whose trafficking from the Golgi to endo-lysosomal compartments drives sequestration of excess Cu and its further excretion from hepatocytes into the bile. Loss of ATP7B function leads to toxic Cu overload in the liver and subsequently in the brain, causing fatal hepatic and neurological abnormalities. The limitations of existing WD therapies call for the development of new therapeutic approaches, which require an amenable animal model system for screening and validation of drugs and molecular targets. To achieve this objective, we generated a mutant Caenorhabditis elegans strain with a substitution of a conserved histidine (H828Q) in the ATP7B ortholog cua-1 corresponding to the most common ATP7B variant (H1069Q) that causes WD. cua-1 mutant animals exhibited very poor resistance to Cu compared to the wild-type strain. This manifested in a strong delay in larval development, a shorter lifespan, impaired motility, oxidative stress pathway activation, and mitochondrial damage. In addition, morphological analysis revealed several neuronal abnormalities in cua-1 mutant animals exposed to Cu. Further investigation suggested that mutant CUA-1 is retained and degraded in the endoplasmic reticulum, similarly to human ATP7B-H1069Q. As a consequence, the mutant protein does not allow animals to counteract Cu toxicity. Notably, pharmacological correctors of ATP7B-H1069Q reduced Cu toxicity in cua-1 mutants indicating that similar pathogenic molecular pathways might be activated by the H/Q substitution and, therefore, targeted for rescue of ATP7B/CUA-1 function. Taken together, our findings suggest that the newly generated cua-1 mutant strain represents an excellent model for Cu toxicity studies in WD.
Assuntos
Degeneração Hepatolenticular , Animais , Humanos , Degeneração Hepatolenticular/genética , Degeneração Hepatolenticular/tratamento farmacológico , Degeneração Hepatolenticular/metabolismo , Cobre/toxicidade , Cobre/metabolismo , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , ATPases Transportadoras de Cobre/genética , ATPases Transportadoras de Cobre/metabolismo , Hepatócitos/metabolismoRESUMO
Endoplasmic reticulum-associated protein degradation (ERAD) is a stringent quality control mechanism through which misfolded, unassembled and some native proteins are targeted for degradation to maintain appropriate cellular and organelle homeostasis. Several in vitro and in vivo ERAD-related studies have provided mechanistic insights into ERAD pathway activation and its consequent events; however, a majority of these have investigated the effect of ERAD substrates and their consequent diseases affecting the degradation process. In this review, we present all reported human single-gene disorders caused by genetic variation in genes that encode ERAD components rather than their substrates. Additionally, after extensive literature survey, we present various genetically manipulated higher cellular and mammalian animal models that lack specific components involved in various stages of the ERAD pathway.
Assuntos
Degradação Associada com o Retículo Endoplasmático , Proteínas , Animais , Humanos , Proteólise , Proteínas/metabolismo , Retículo Endoplasmático/metabolismo , Fenótipo , Mamíferos/genética , Mamíferos/metabolismoRESUMO
Glutathione (GSH) is a highly abundant tripeptide thiol that performs diverse protective and biosynthetic functions in cells. While changes in GSH availability are associated with inborn errors of metabolism, cancer, and neurodegenerative disorders, studying the limiting role of GSH in physiology and disease has been challenging due to its tight regulation. To address this, we generated cell and mouse models that express a bifunctional glutathione-synthesizing enzyme from Streptococcus thermophilus (GshF), which possesses both glutamate-cysteine ligase and glutathione synthase activities. GshF expression allows efficient production of GSH in the cytosol and mitochondria and prevents cell death in response to GSH depletion, but not ferroptosis induction, indicating that GSH is not a limiting factor under lipid peroxidation. CRISPR screens using engineered enzymes further revealed genes required for cell proliferation under cellular and mitochondrial GSH depletion. Among these, we identified the glutamate-cysteine ligase modifier subunit, GCLM, as a requirement for cellular sensitivity to buthionine sulfoximine, a glutathione synthesis inhibitor. Finally, GshF expression in mice is embryonically lethal but sustains postnatal viability when restricted to adulthood. Overall, our work identifies a conditional mouse model to investigate the limiting role of GSH in physiology and disease.
Assuntos
Glutamato-Cisteína Ligase , Glutationa , Animais , Camundongos , Butionina Sulfoximina/farmacologia , Modelos Animais de Doenças , Glutamato-Cisteína Ligase/genética , Glutamato-Cisteína Ligase/metabolismo , Glutationa/metabolismo , Linhagem Celular Tumoral , HumanosRESUMO
BACKGROUND & AIMS: Hepatitis E virus (HEV), primarily genotype 1 (HEV-1), causes approximately 20.1 million infections, 44,000 deaths, and 3000 stillbirths annually. Current evidence indicates that HEV-1 is only transmitted in humans. Here, we evaluated whether Mongolian gerbils can serve as animal models for HEV-1 infection. METHODS: Mongolian gerbils were used for HEV-1 and hepatitis E virus genotype 3 infection experiments. HEV infection parameters, including detection of HEV RNA and HEV antigen, liver function assessment, and histopathology, were evaluated. RESULTS: We adapted a clinical isolate of HEV-1 for Mongolian gerbils by serial passaging in feces of aged male gerbils. The gerbil-adapted strain obtained at passage 3 induced a robust, acute HEV infection, characterized by stable fecal virus shedding, elevated liver enzymes, histopathologic changes in the liver, and seroconversion to anti-HEV. An infectious complementary DNA clone of the adapted virus was generated. HEV-1-infected pregnant gerbils showed a high rate of maternal mortality and vertical transmission. HEV RNA or antigens were detected in the liver, kidney, intestine, placenta, testis, and fetus liver. Liver and placental transcriptomic analyses indicated activation of host immunity. Tacrolimus prolonged HEV-1 infection, whereas ribavirin cleared infection. The protective efficacy of a licensed HEV vaccine was validated using this model. CONCLUSIONS: HEV-1 efficiently infected Mongolian gerbils. This HEV-1 infection model will be valuable for investigating hepatitis E immunopathogenesis and evaluating vaccines and antivirals against HEV.
Assuntos
Modelos Animais de Doenças , Genótipo , Gerbillinae , Vírus da Hepatite E , Hepatite E , Imunocompetência , Fígado , RNA Viral , Animais , Vírus da Hepatite E/genética , Vírus da Hepatite E/patogenicidade , Vírus da Hepatite E/imunologia , Hepatite E/virologia , Hepatite E/imunologia , Hepatite E/transmissão , Masculino , Feminino , RNA Viral/isolamento & purificação , RNA Viral/análise , Fígado/virologia , Fígado/patologia , Fezes/virologia , Gravidez , Transmissão Vertical de Doenças Infecciosas , Antivirais/uso terapêutico , Antivirais/farmacologia , Eliminação de Partículas Virais , Ribavirina/uso terapêutico , Ribavirina/farmacologia , Complicações Infecciosas na Gravidez/virologia , Complicações Infecciosas na Gravidez/imunologiaRESUMO
Animal models of authentic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection require operation in biosafety level 3 (BSL-3) containment. In the present study, we established a mouse model employing a single-cycle infectious virus replicon particle (VRP) system of SARS-CoV-2 that can be safely handled in BSL-2 laboratories. The VRP [ΔS-VRP(G)-Luc] contains a SARS-CoV-2 genome in which the spike gene was replaced by a firefly luciferase (Fluc) reporter gene (Rep-Luci), and incorporates the vesicular stomatitis virus glycoprotein on the surface. Intranasal inoculation of ΔS-VRP(G)-Luc can successfully transduce the Rep-Luci genome into mouse lungs, initiating self-replication of Rep-Luci and, accordingly, inducing acute lung injury mimicking the authentic SARS-CoV-2 pathology. In addition, the reporter Fluc expression can be monitored using a bioluminescence imaging approach, allowing a rapid and convenient determination of viral replication in ΔS-VRP(G)-Luc-infected mouse lungs. Upon treatment with an approved anti-SARS-CoV-2 drug, VV116, the viral replication in infected mouse lungs was significantly reduced, suggesting that the animal model is feasible for antiviral evaluation. In summary, we have developed a BSL-2-compliant mouse model of SARS-CoV-2 infection, providing an advanced approach to study aspects of the viral pathogenesis, viral-host interactions, as well as the efficacy of antiviral therapeutics in the future.IMPORTANCESevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is highly contagious and pathogenic in humans; thus, research on authentic SARS-CoV-2 has been restricted to biosafety level 3 (BSL-3) laboratories. However, due to the scarcity of BSL-3 facilities and trained personnel, the participation of a broad scientific community in SARS-CoV-2 research had been greatly limited, hindering the advancement of our understanding on the basic virology as well as the urgently necessitated drug development. Previously, our colleagues Jin et al. had generated a SARS-CoV-2 replicon by replacing the essential spike gene in the viral genome with a Fluc reporter (Rep-Luci), which can be safely operated under BSL-2 conditions. By incorporating the Rep-Luci into viral replicon particles carrying vesicular stomatitis virus glycoprotein on their surface, and via intranasal inoculation, we successfully transduced the Rep-Luci into mouse lungs, developing a mouse model mimicking SARS-CoV-2 infection. Our model can serve as a useful platform for SARS-CoV-2 pathological studies and antiviral evaluation under BSL2 containment.
Assuntos
Antivirais , COVID-19 , Modelos Animais de Doenças , Genes Reporter , SARS-CoV-2 , Replicação Viral , Animais , SARS-CoV-2/fisiologia , SARS-CoV-2/genética , Camundongos , COVID-19/virologia , Antivirais/farmacologia , Antivirais/uso terapêutico , Humanos , Pulmão/virologia , Pulmão/patologia , Betacoronavirus/fisiologia , Betacoronavirus/genética , Pneumonia Viral/virologia , Infecções por Coronavirus/virologia , Contenção de Riscos Biológicos , Pandemias , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Feminino , Camundongos Endogâmicos BALB C , Chlorocebus aethiops , Replicon , Células Vero , Luciferases de Vaga-Lume/genética , Luciferases de Vaga-Lume/metabolismoRESUMO
Autoimmune diseases often arise from conditions where the immune system is compromised. While lymphopenia-induced proliferation (LIP) is crucial for immune system development and maturation, it is also caused by environmental insults, such as infection, and becomes a risk factor for autoimmunity in adults. We used Dsg3H1 TCR transgenic mice, whose T cells are designed to recognize desmogrein-3, a skin antigen, to explore the impact of lymphopenia on post-thymic tolerance. Dsg3H1 mice are known to delete the most highly autoreactive T cells in the thymus, and develop only subtle immune-mediated pathology in the steady state. However, we found that transient lymphopenia induced by total body irradiation (TBI) or cyclophosphamide (CY) results in massive dermatitis in Dsg3H1 mice. The symptoms included expansion and development of self-reactive T cells, their differentiation into CD44high IL-17-producing helper T cells, and severe neutrophilic inflammation. Repopulation of FOXP3+ T regulatory cells after lymphopenia normally occurred, suggesting escape of skin-reactive conventional T cells from control by regulatory T cells. Furthermore, we found that a depletion of the intestinal microbiota by antibiotics prevents CY-induced dermatitis, indicating roles of the commensal intestinal microbiota in LIP and Th17 development in vivo. The current data suggested that post-thymic tolerance of Dsg3H1 mice is established on a fragile balance in the lymphoreplete immune environment and broken by the interplay between lymphopenia and intestinal microbiota. The dynamic phenotypes observed in Dsg3H1 mice prompt a re-evaluation of opportunistic lymphopenia together with the microbiota as pivotal environmental factors, impacting individuals with genetic predispositions for autoimmune diseases.
Assuntos
Microbioma Gastrointestinal , Tolerância Imunológica , Linfopenia , Camundongos Transgênicos , Animais , Camundongos , Microbioma Gastrointestinal/imunologia , Linfopenia/imunologia , Tolerância Imunológica/imunologia , Pele/imunologia , Pele/microbiologia , Pele/patologia , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Timo/imunologia , Camundongos Endogâmicos C57BLRESUMO
Bat sarbecovirus BANAL-236 is highly related to SARS-CoV-2 and infects human cells, albeit lacking the furin cleavage site in its spike protein. BANAL-236 replicates efficiently and pauci-symptomatically in humanized mice and in macaques, where its tropism is enteric, strongly differing from that of SARS-CoV-2. BANAL-236 infection leads to protection against superinfection by a virulent strain. We find no evidence of antibodies recognizing bat sarbecoviruses in populations in close contact with bats in which the virus was identified, indicating that such spillover infections, if they occur, are rare. Six passages in humanized mice or in human intestinal cells, mimicking putative early spillover events, select adaptive mutations without appearance of a furin cleavage site and no change in virulence. Therefore, acquisition of a furin site in the spike protein is likely a pre-spillover event that did not occur upon replication of a SARS-CoV-2-like bat virus in humans or other animals. Other hypotheses regarding the origin of the SARS-CoV-2 should therefore be evaluated, including the presence of sarbecoviruses carrying a spike with a furin cleavage site in bats.
Assuntos
COVID-19 , Humanos , Animais , Camundongos , SARS-CoV-2 , Furina/genética , Furina/metabolismo , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , MutaçãoRESUMO
Neuromyelitis optica spectrum disorder (NMOSD) is a CNS autoimmune inflammatory disease mediated by T helper 17 (Th17) and antibody responses to the water channel protein, aquaporin 4 (AQP4), and associated with astrocytopathy, demyelination and axonal loss. Knowledge about disease pathogenesis is limited and the search for new therapies impeded by the absence of a reliable animal model. In our work, we determined that NMOSD is characterized by decreased IFN-γ receptor signalling and that IFN-γ depletion in AQP4201-220-immunized C57BL/6 mice results in severe clinical disease resembling human NMOSD. Pathologically, the disease causes autoimmune astrocytic and CNS injury secondary to cellular and humoral inflammation. Immunologically, the absence of IFN-γ allows for increased expression of IL-6 in B cells and activation of Th17 cells, and generation of a robust autoimmune inflammatory response. Consistent with NMOSD, the experimental disease is exacerbated by administration of IFN-ß, whereas repletion of IFN-γ, as well as therapeutic targeting of IL-17A, IL-6R and B cells, ameliorates it. We also demonstrate that immune tolerization with AQP4201-220-coupled poly(lactic-co-glycolic acid) nanoparticles could both prevent and effectively treat the disease. Our findings enhance the understanding of NMOSD pathogenesis and provide a platform for the development of immune tolerance-based therapies, avoiding the limitations of the current immunosuppressive therapies.
Assuntos
Neuromielite Óptica , Humanos , Animais , Camundongos , Neuromielite Óptica/patologia , Aquaporina 4 , Interferon gama/metabolismo , Camundongos Endogâmicos C57BL , Linfócitos B , Autoanticorpos/metabolismoRESUMO
The clinical manifestations of sporadic amyotrophic lateral sclerosis (ALS) vary widely. However, the current classification of ALS is mainly based on clinical presentations, while the roles of electrophysiological and biomedical biomarkers remain limited. Herein, we investigated a group of patients with sporadic ALS and an ALS mouse model with superoxide dismutase 1 (SOD1)/G93A transgenes using nerve excitability tests (NET) to investigate axonal membrane properties and chemical precipitation, followed by enzyme-linked immunosorbent assay analysis to measure plasma misfolded protein levels. Six of 19 patients (31.6%) with sporadic ALS had elevated plasma misfolded SOD1 protein levels. In sporadic ALS patients, only those with elevated misfolded SOD1 protein levels showed an increased inward rectification in the current-threshold (I/V) curve and an increased threshold reduction in the hyperpolarizing threshold electrotonus (TE) in the NET study. Two familial ALS patients with SOD1 mutations also exhibited similar electrophysiological patterns of NET. For patients with sporadic ALS showing significantly increased inward rectification in the I/V curve, we noted an elevation in plasma misfolded SOD1 level, but not in total SOD1, misfolded C9orf72, or misfolded phosphorylated TDP43 levels. Computer simulations demonstrated that the aforementioned axonal excitability changes are likely associated with an increase in hyperpolarization-activated cyclic nucleotide-gated (HCN) current. In SOD1/G93A mice, NET also showed an increased inward rectification in the I/V curve, which could be reversed by a single injection of the HCN channel blocker, ZD7288. Daily treatment of SOD1/G93A mice with ZD7288 partially prevented the early motor function decline and spinal motor neuron death. In summary, sporadic ALS patients with elevated plasma misfolded SOD1 exhibited similar patterns of motor axonal excitability changes as familial ALS patients and ALS mice with mutant SOD1 genes, suggesting the existence of SOD1-associated sporadic ALS. The observed NET pattern of increased inward rectification in the I/V curve was attributable to an elevation in the HCN current in SOD1-associated ALS.
RESUMO
Human papillomavirus (HPV) 16 and 18 infections are related to many human cancers. Despite several preventive vaccines for high-risk (hr) HPVs, there is still an urgent need to develop therapeutic HPV vaccines for targeting pre-existing hrHPV infections and lesions. In this study, we developed a lipid nanoparticle (LNP)-formulated mRNA-based HPV therapeutic vaccine (mHTV)-03E2, simultaneously targeting the E2/E6/E7 of both HPV16 and HPV18. mHTV-03E2 dramatically induced antigen-specific cellular immune responses, leading to significant CD8+ T cell infiltration and cytotoxicity in TC-1 tumors derived from primary lung epithelial cells of C57BL/6 mice expressing HPV E6/E7 antigens, mediated significant tumor regression, and prolonged animal survival, in a dose-dependent manner. We further demonstrated significant T cell immunity against HPV16/18 E6/E7 antigens for up to 4 months post-vaccination in immunological and distant tumor rechallenging experiments, suggesting robust memory T cell immunity against relapse. Finally, mHTV-03E2 synergized with immune checkpoint blockade to inhibit tumor growth and extend animal survival, indicating the potential in combination therapy. We conclude that mHTV-03E2 is an excellent candidate therapeutic mRNA vaccine for treating malignancies caused by HPV16 or HPV18 infections.
Assuntos
Proteínas Oncogênicas Virais , Infecções por Papillomavirus , Vacinas contra Papillomavirus , RNA Mensageiro , Animais , Camundongos , Vacinas contra Papillomavirus/imunologia , Humanos , Infecções por Papillomavirus/imunologia , Infecções por Papillomavirus/virologia , Infecções por Papillomavirus/terapia , Infecções por Papillomavirus/prevenção & controle , Feminino , Proteínas Oncogênicas Virais/imunologia , Proteínas Oncogênicas Virais/genética , RNA Mensageiro/genética , RNA Mensageiro/imunologia , Nanopartículas/química , Papillomavirus Humano 16/imunologia , Papillomavirus Humano 16/genética , Camundongos Endogâmicos C57BL , Papillomavirus Humano 18/imunologia , Papillomavirus Humano 18/genética , Proteínas E7 de Papillomavirus/imunologia , Proteínas E7 de Papillomavirus/genética , Vacinas Anticâncer/imunologia , Vacinas Anticâncer/administração & dosagem , Linhagem Celular Tumoral , Modelos Animais de Doenças , Linfócitos T CD8-Positivos/imunologia , Proteínas Repressoras/imunologia , Proteínas Repressoras/genética , Proteínas de Ligação a DNA , LipossomosRESUMO
Neurological disorders pose a challenge for targeted therapy due to restricted access of therapeutic agents to the central nervous system (CNS). Current methods are limited by procedure-related risks, invasiveness, and insufficient CNS biodistribution. A novel percutaneous transvenous technology, currently in clinical trials for communicating hydrocephalus, offers a minimally invasive approach by providing endovascular access to the cerebrospinal fluid-filled cerebellopontine angle (CPA) cistern. We hypothesized that drug delivery to the CPA cistern could yield widespread CNS distribution. Using an ovine model, we compared the biodistribution of scAAV9-CB-GFP following CPA cistern infusion with previously reported cisterna magna (CM) administration. Targeting both the CPA cistern and CM in sheep, we employed a lumbar spine-inserted microcatheter under fluoroscopy. CPA delivery of AAV9 demonstrated biodistribution and transduction in the cerebral cortices, striatum, thalamus, midbrain, cerebellum, and spinal cord, with minor liver distribution comparable to CM. The favorable safety profile in humans with hydrocephalus suggests that percutaneous endovascular injection into the CPA could offer a clinically safer and minimally invasive delivery system for CNS gene and cell-based therapies.
Assuntos
Ângulo Cerebelopontino , Dependovirus , Vetores Genéticos , Proteínas de Fluorescência Verde , Animais , Dependovirus/genética , Vetores Genéticos/administração & dosagem , Vetores Genéticos/farmacocinética , Vetores Genéticos/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Ovinos , Distribuição Tecidual , Procedimentos Endovasculares/métodos , Humanos , Sistema Nervoso Central/metabolismo , Cisterna Magna/metabolismo , Terapia Genética/métodos , Técnicas de Transferência de GenesRESUMO
X chromosome inactivation (XCI) is a process that equalizes the expression of X-linked genes between males and females. It relies on Xist, continuously expressed in somatic cells during XCI maintenance. However, how Xist impacts XCI maintenance and its functional motifs remain unclear. In this study, we conducted a comprehensive analysis of Xist, using rabbits as an ideal non-primate model. Homozygous knockout of exon 1, exon 6, and repeat A in female rabbits resulted in embryonic lethality. However, X∆ReAX females, with intact X chromosome expressing Xist, showed no abnormalities. Interestingly, there were no significant differences between females with homozygous knockout of exons 2-5 and wild-type rabbits, suggesting that exons 2, 3, 4, and 5 are less important for XCI. These findings provide evolutionary insights into Xist function.
Assuntos
RNA Longo não Codificante , Inativação do Cromossomo X , Humanos , Masculino , Animais , Coelhos , Feminino , Inativação do Cromossomo X/genética , RNA Longo não Codificante/genética , Cromossomos Humanos X , Cromossomo X/genética , Éxons/genéticaRESUMO
Rationale: Pneumonia is a frequent and feared complication in intubated critically ill patients. Tissue concentrations of antimicrobial drugs need to be sufficiently high to treat the infection and also prevent development of bacterial resistance. It is uncertain whether pulmonary inflammation and injury affect antimicrobial drug penetration into lung tissue.Objectives: To determine and compare tissue and BAL fluid concentrations of ceftaroline fosamil and linezolid in a model of unilateral acute lung injury in pigs and to evaluate whether dose adjustment is necessary to reach sufficient antimicrobial concentrations in injured lung tissue.Methods: After induction of unilateral acute lung injury, ceftaroline fosamil and linezolid were administered intravenously. Drug concentrations were measured in lung tissue through microdialysis and in blood and BAL fluid samples during the following 8 hours. The primary endpoint was the tissue concentration area under the concentration curve in the first 8 hours (AUC0-8 h) of the two antimicrobial drugs.Measurements and Main Results: In 10 pigs, antimicrobial drug concentrations were higher in inflamed and injured lung tissue compared with those in uninflamed and uninjured lung tissue (median ceftaroline fosamil AUC0-8 h [and interquartile range] = 26.7 mg â h â L-1 [19.7-39.0] vs. 16.0 mg â h â L-1 [13.6-19.9], P = 0.02; median linezolid AUC0-8 h 76.0 mg â h â L-1 [68.1-96.0] vs. 54.6 mg â h â L-1 [42.7-60.9], P = 0.01), resulting in a longer time above the minimal inhibitory concentration and in higher peak concentrations and dialysate/plasma ratios. Penetration into BAL fluid was excellent for both antimicrobials, but without left-to-right differences (ceftaroline fosamil, P = 0.78; linezolid, P = 1.00).Conclusions: Tissue penetration of two commonly used antimicrobial drugs for pneumonia is enhanced by early lung tissue inflammation and injury, resulting in longer times above the minimal inhibitory concentration. Thus, lung tissue inflammation ameliorates antimicrobial drug penetration during the acute phase.
Assuntos
Lesão Pulmonar Aguda , Anti-Infecciosos , Pneumonia , Humanos , Animais , Suínos , Linezolida/uso terapêutico , Antibacterianos/efeitos adversos , Anti-Infecciosos/uso terapêutico , Ceftarolina , Pneumonia/tratamento farmacológico , Pneumonia/induzido quimicamente , Inflamação/tratamento farmacológico , Inflamação/induzido quimicamente , Pulmão , Lesão Pulmonar Aguda/tratamento farmacológico , Lesão Pulmonar Aguda/induzido quimicamenteRESUMO
Despite excellent vaccines, resurgent outbreaks of hepatitis A have caused thousands of hospitalizations and hundreds of deaths within the United States in recent years. There is no effective antiviral therapy for hepatitis A, and many aspects of the hepatitis A virus (HAV) replication cycle remain to be elucidated. Replication requires the zinc finger protein ZCCHC14 and noncanonical TENT4 poly(A) polymerases with which it associates, but the underlying mechanism is unknown. Here, we show that ZCCHC14 and TENT4A/B are required for viral RNA synthesis following translation of the viral genome in infected cells. Cross-linking immunoprecipitation sequencing (CLIP-seq) experiments revealed that ZCCHC14 binds a small stem-loop in the HAV 5' untranslated RNA possessing a Smaug recognition-like pentaloop to which it recruits TENT4. TENT4 polymerases lengthen and stabilize the 3' poly(A) tails of some cellular and viral mRNAs, but the chemical inhibition of TENT4A/B with the dihydroquinolizinone RG7834 had no impact on the length of the HAV 3' poly(A) tail, stability of HAV RNA, or cap-independent translation of the viral genome. By contrast, RG7834 inhibited the incorporation of 5-ethynyl uridine into nascent HAV RNA, indicating that TENT4A/B function in viral RNA synthesis. Consistent with potent in vitro antiviral activity against HAV (IC50 6.11 nM), orally administered RG7834 completely blocked HAV infection in Ifnar1-/- mice, and sharply reduced serum alanine aminotransferase activities, hepatocyte apoptosis, and intrahepatic inflammatory cell infiltrates in mice with acute hepatitis A. These results reveal requirements for ZCCHC14-TENT4A/B in hepatovirus RNA synthesis, and suggest that TENT4A/B inhibitors may be useful for preventing or treating hepatitis A in humans.
Assuntos
Proteínas Cromossômicas não Histona , DNA Polimerase Dirigida por DNA , Vírus da Hepatite A , Hepatite A , Proteínas Intrinsicamente Desordenadas , RNA Nucleotidiltransferases , RNA Viral , Replicação Viral , Animais , Antivirais/farmacologia , Antivirais/uso terapêutico , Proteínas Cromossômicas não Histona/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Hepatite A/tratamento farmacológico , Hepatite A/metabolismo , Hepatite A/virologia , Vírus da Hepatite A/efeitos dos fármacos , Vírus da Hepatite A/genética , Vírus da Hepatite A/fisiologia , Humanos , Proteínas Intrinsicamente Desordenadas/metabolismo , Camundongos , Camundongos Mutantes , RNA Nucleotidiltransferases/metabolismo , RNA Viral/biossíntese , RNA Viral/genética , Receptor de Interferon alfa e beta/genética , Replicação Viral/efeitos dos fármacosRESUMO
SignificanceCanine models of inherited retinal diseases have helped advance adeno-associated virus (AAV)-based gene therapies targeting specific cells in the outer retina for treating blinding diseases in patients. However, therapeutic targeting of diseases such as congenital stationary night blindness (CSNB) that exhibit defects in ON-bipolar cells (ON-BCs) of the midretina remains underdeveloped. Using a leucine-rich repeat, immunoglobulin-like and transmembrane domain 3 (LRIT3) mutant canine model of CSNB exhibiting ON-BC dysfunction, we tested the ability of cell-specific AAV capsids and promotors to specifically target ON-BCs for gene delivery. Subretinal injection of one vector demonstrated safety and efficacy with robust and stable rescue of electroretinography signals and night vision up to 1 y, paving the way for clinical trials in patients.
Assuntos
Doenças Genéticas Ligadas ao Cromossomo X , Cegueira Noturna , Animais , Dependovirus/genética , Cães , Eletrorretinografia , Oftalmopatias Hereditárias , Doenças Genéticas Ligadas ao Cromossomo X/genética , Doenças Genéticas Ligadas ao Cromossomo X/terapia , Terapia Genética , Humanos , Proteínas de Membrana/genética , Miopia , Cegueira Noturna/genética , Cegueira Noturna/terapiaRESUMO
The most recent Sudan virus (SUDV) outbreak in Uganda was first detected in September 2022 and resulted in 164 laboratory-confirmed cases and 77 deaths. There are no approved vaccines against SUDV. Here, we investigated the protective efficacy of ChAdOx1-biEBOV in cynomolgus macaques using a prime or a prime-boost regimen. ChAdOx1-biEBOV is a replication-deficient simian adenovirus vector encoding SUDV and Ebola virus (EBOV) glycoproteins (GPs). Intramuscular vaccination induced SUDV and EBOV GP-specific IgG responses and neutralizing antibodies. Upon challenge with SUDV, vaccinated animals showed signs of disease like those observed in control animals, and no difference in survival outcomes were measured among all three groups. Viral load in blood samples and in tissue samples obtained after necropsy were not significantly different between groups. Overall, this study highlights the importance of evaluating vaccines in multiple animal models and demonstrates the importance of understanding protective efficacy in both animal models and human hosts.