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Genetically modified oncolytic adenoviruses have been proposed as a vehicle for cancer therapy. However, several concerns, such as toxicity to normal cells and organs, lack of suitable cell surface receptors to allow viral entry to the desired cell type(s), and activation of both innate and adaptive immune systems in patients, restrict the successful clinical application of adenoviral-mediated cancer gene therapy. Successful virotherapy will require efficient transductional and transcriptional targeting to enhance therapeutic efficacy by ensuring targeted adenoviral infection, replication, and/or therapeutic transgene expression. Targeted modification of viral components, such as viral capsid, fiber knob, and the insertion of transgenes for expression, are prerequisites for the necessary transductional and transcriptional targeting of adenovirus. However, the conventional approach to modify the adenoviral genome is complex, time consuming, and expensive. It is dependent on the presence of unique restriction enzyme sites that may or may not be present in the target location. Clustered regularly interspaced short palindromic repeat (CRISPR) along with the RNA-guided nuclease Cas9 (CRISPR/Cas9) is one of the most powerful tools that has been adopted for precise genome editing in a variety of cells and organisms. However, the ability of the CRISPR/Cas9 system to precisely and efficiently make genetic modification, as well as introduce gene replacements, in adenoviral genomes, remains essentially unknown. Herein the ability of in vitro CRISPR/CAS9-mediated editing of the canine adenovirus type 2 (CAV2) genome to promote targeted modification of the viral genome was assessed. To demonstrate the feasibility of this goal, CRISPR/Cas9 has been used to successfully insert the RFP (red fluorescent protein) reporter construct into the CAV2 genome. Initial results demonstrated high efficiency and accuracy for in vitro CRISPR-mediated editing of the large CAV2 genome. Furthermore, this application was expanded, using multiple guide RNAs, to conduct gene replacement in the CAV2 genome by substituting a portion of the E3 gene with a construct designed to express a single chain antibody to canine PD-1. Thus, this work provides a significantly improved and efficient method for targeted editing of adenoviruses to generate altered and potentially therapeutic viral genomes in the shortest possible time.
Asunto(s)
Adenovirus Caninos/genética , Edición Génica , Animales , Proteína 9 Asociada a CRISPR , Sistemas CRISPR-Cas , Línea Celular Tumoral , Perros , Genoma Viral , Viroterapia Oncolítica , Reparación del ADN por RecombinaciónRESUMEN
Alzheimer's disease (AD) and cardiovascular disease (CVD) are pathologies that are characterized by common signatures of vascular dysfunction and chronic inflammation that are accelerated with aging. Importantly, epidemiological studies report an independent interaction between AD and CVD and data suggest that chronic inflammation in CVD may accelerate AD development. Atherosclerosis affects most large to medium sized arteries including those supplying the cerebral circulation. Vascular dysfunction caused by atherosclerosis results in blood brain barrier breakdown, inflammation, an impaired clearance of amyloid-beta (Aß), and finally ends with neurovascular dysfunction. Numerous data indicate that innate and adaptive immune responses shape atherogenesis and increasing evidence suggests an implication of the immune response in AD progression. Currently, mechanisms by which these two diseases are interconnected with each other are not well-defined. In this review, we discuss the recent advances in our understanding of the intertwined role of the immune response in atherosclerosis and AD and the implications of these findings for human health.
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Atherosclerosis is a chronic inflammatory disease of the vascular system that is characterized by the deposition of modified lipoproteins, accumulation of immune cells, and formation of fibrous tissue within the vessel wall. The disease occurs in vessels throughout the body and affects the functions of almost all organs including the lymphoid system, bone marrow, heart, brain, pancreas, adipose tissue, liver, kidneys, and gastrointestinal tract. Atherosclerosis and associated factors influence these tissues via the modulation of local vascular functions, induction of cholesterol-associated pathologies, and regulation of local immune responses. In this review, we discuss how atherosclerosis interferers with functions of different organs via several common pathways and how the disturbance of immunity in atherosclerosis can result in disease-provoking dysfunctions in multiple tissues. Our growing appreciation of the implication of atherosclerosis and associated microenvironmental conditions in the multi-organ pathology promises to influence our understanding of CVD-associated disease pathologies and to provide new therapeutic opportunities.
Asunto(s)
Aterosclerosis , Tejido Adiposo , Aterosclerosis/etiología , Tracto Gastrointestinal , Humanos , Riñón , HígadoRESUMEN
Delay in cancer diagnosis often results in metastasis and an inability to successfully treat the tumor. The use of broadly cancer-specific biomarkers at an early stage may improve cancer treatment and staging. This study has explored circulatory exosomal miRNAs as potential diagnostic biomarkers to identify cancer patients. Secretory exosomal miRNAs were isolated from 13 canine cancer cell lines (lymphoma, mast cell tumor, histiocytic cell line, osteosarcoma, melanoma, and breast tumor) and were sequenced by Next-Generation sequencing (NGS). We have identified 6 miRNAs (cfa-miR-9, -1841, -1306, -345, -132, and -26b) by NGS that were elevated in all cancer cell types. The miRNAs identified by NGS were then examined by Q-RT-PCR. The PCR data demonstrated similar expression patterns to those seen with NGS but provided fold differences that were much lower than those seen for NGS. Cfa-miR-9 was found to be the most consistently elevated miRNA in NGS and PCR, making it the most likely miRNA to prove diagnostic. In this study, we have demonstrated that it is possible to identify exosomal miRNAs with elevated secretion across multiple tumor types that could be used as circulatory diagnostic biomarkers for liquid biopsy in the future.
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Sensitivity to allergenic fungi (Alternaria alternata) is associated with acute, severe asthma attacks. Antigen presenting cells (APCs) in the lung sense environmental perturbations that induce cellular stress and metabolic changes and are critical for allergic airway inflammation. However, the mechanisms underlying such environmental sensing by APCs in the lung remains unclear. Here we show that acute Alternaria challenge rapidly induces neutrophil accumulation in airways, and alter expressions of Pyruvate Kinase (PKM2) and hypoxia-inducible factor -1α (Hif-1α) that correlates with proinflammatory mediator release. Blockade of IL33 signaling in vivo led to reduce oxidative stress and glycolysis in lung APCs. Lung-specific ablation of CD11c+ cells abrogates Alternaria-induced neutrophil accumulation and inflammation. Furthermore, administration of Alternaria into the airways stimulated APCs and elevate the expression of Glut-1. Mechanistically, we establish that PKM2 is a critical modulator of lung APC activation in Alternaria-induced acute inflammation. Allosteric activation of PKM2 by a small molecule ML265 or siRNA-mediated knock down correlated negatively with glycolysis and activation of APCs. These results collectively demonstrates that PKM2-mediated glycolytic reprogramming by fungal allergen Alternaria influences lung APC activation, thereby promotes acute airway inflammation. Our data support a model in which Alternaria sensitization in airways induce a circuitry of glycolysis and PKM2 regulation that confers an acute activation of APCs in the lung, whose targeting might represent a strategy for asthma treatment.
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Alternaria/inmunología , Alternariosis/metabolismo , Alternariosis/microbiología , Células Presentadoras de Antígenos/inmunología , Células Presentadoras de Antígenos/metabolismo , Piruvato Quinasa/metabolismo , Hipersensibilidad Respiratoria/metabolismo , Hipersensibilidad Respiratoria/microbiología , Alérgenos/inmunología , Animales , Asma/etiología , Asma/metabolismo , Asma/patología , Biomarcadores , Citocinas/metabolismo , Modelos Animales de Enfermedad , Glucosa/metabolismo , Glucólisis , Interacciones Huésped-Patógeno/genética , Interacciones Huésped-Patógeno/inmunología , Inmunofenotipificación , Mediadores de Inflamación/metabolismo , Pulmón/inmunología , Pulmón/metabolismo , Pulmón/patología , Ratones , Ratones Noqueados , Ratones Transgénicos , Piruvato Quinasa/genética , Especies Reactivas de Oxígeno/metabolismo , Hipersensibilidad Respiratoria/patologíaRESUMEN
Proliferation of dendritic cell (DC)-restricted progenitor cells in bone marrow compartment is tightly regulated at steady state and responds to multiple tissue-specific triggers during disturbed homeostasis such as obesity. DCs in the lung stem from a rapidly dividing DC-restricted progenitor cells and are effective at generating adaptive immune responses in allergic airway inflammation. Precisely, how DC-restricted progenitor expansion and differentiation are influenced by airway inflammation to maintain constant supply of myeloid DCs is poorly understood. Here we show that a high fat diet (HFD) induces oxidative stress and accelerates the expansion of DC- restricted progenitor cells in bone marrow and correlates with persistent induction of p38 mitogen activated protein kinase (MAPK), which is blocked with a selective p38α/ß MAPK inhibitor. Mice fed a HFD and sensitized to inhaled allergen house dust mite (HDM) led to alterations of DC- restricted progenitor cells that were characterized by increased expansion and seeding of lung DCs in airway inflammation. Mechanistically, we establish that the expansion induced by HFD dysregulates the expression of a disintegrin and metallopeptidase domain 17 (Adam17) and is required for p38 MAPK activation in DC-restricted progenitors. These results demonstrates that obesity produces persistent changes in DC precursors and that elevation of Adam17 expression is tightly coupled to p38 MAPK and is a key driver of proliferation. Altogether, these data provide phenotypic and mechanistic insight into dendritic cell supply chain in obesity-associated airway inflammation.