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Conditions affecting the brain are the second leading cause of death globally. One of the main challenges for drugs targeting brain diseases is passing the blood-brain barrier (BBB). Here, the effectiveness of mesoporous silica nanostars (MSiNSs) with two different spike lengths to cross an in vitro BBB multicellular model was evaluated and compared to spherical nanoparticles (MSiNP). A modified sol-gel single-micelle epitaxial growth was used to produce MSiNS, which showed no cytotoxicity or immunogenicity at concentrations of up to 1 µg mL-1 in peripheral blood mononuclear and neuronal cells. The nanostar MSiNS effectively penetrated the BBB model after 24 h, and MSiNS-1 with a shorter spike length (9 ± 2 nm) crossed the in vitro BBB model more rapidly than the MSiNS-2 with longer spikes (18 ± 4 nm) or spherical MSiNP at 96 h, which accumulated in the apical and basolateral sides, respectively. Molecular dynamic simulations illustrated an increase in configurational flexibility of the lipid bilayer during contact with the MSiNS, resulting in wrapping, whereas the MSiNP suppressed membrane fluctuations. This work advances an effective brain drug delivery system based on virus-like shaped MSiNS for the treatment of different brain diseases and a mechanism for their interaction with lipid bilayers.
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Barrera Hematoencefálica , Dióxido de Silicio , Dióxido de Silicio/química , Barrera Hematoencefálica/metabolismo , Barrera Hematoencefálica/efectos de los fármacos , Humanos , Porosidad , Nanopartículas/química , Sistemas de Liberación de Medicamentos , Simulación de Dinámica Molecular , Portadores de Fármacos/química , Transporte Biológico , Animales , Membrana Dobles de Lípidos/química , Membrana Dobles de Lípidos/metabolismoRESUMEN
Disruption of the lung endothelial-epithelial cell barrier following respiratory virus infection causes cell and fluid accumulation in the air spaces and compromises vital gas exchange function1. Endothelial dysfunction can exacerbate tissue damage2,3, yet it is unclear whether the lung endothelium promotes host resistance against viral pathogens. Here we show that the environmental sensor aryl hydrocarbon receptor (AHR) is highly active in lung endothelial cells and protects against influenza-induced lung vascular leakage. Loss of AHR in endothelia exacerbates lung damage and promotes the infiltration of red blood cells and leukocytes into alveolar air spaces. Moreover, barrier protection is compromised and host susceptibility to secondary bacterial infections is increased when endothelial AHR is missing. AHR engages tissue-protective transcriptional networks in endothelia, including the vasoactive apelin-APJ peptide system4, to prevent a dysplastic and apoptotic response in airway epithelial cells. Finally, we show that protective AHR signalling in lung endothelial cells is dampened by the infection itself. Maintenance of protective AHR function requires a diet enriched in naturally occurring AHR ligands, which activate disease tolerance pathways in lung endothelia to prevent tissue damage. Our findings demonstrate the importance of endothelial function in lung barrier immunity. We identify a gut-lung axis that affects lung damage following encounters with viral pathogens, linking dietary composition and intake to host fitness and inter-individual variations in disease outcome.
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Células Endoteliales , Pulmón , Infecciones por Orthomyxoviridae , Receptores de Hidrocarburo de Aril , Animales , Humanos , Ratones , Apelina/metabolismo , Dieta , Células Endoteliales/metabolismo , Endotelio/citología , Endotelio/metabolismo , Células Epiteliales/metabolismo , Eritrocitos/metabolismo , Gripe Humana/inmunología , Gripe Humana/metabolismo , Intestinos/metabolismo , Leucocitos/metabolismo , Ligandos , Pulmón/inmunología , Pulmón/metabolismo , Infecciones por Orthomyxoviridae/inmunología , Infecciones por Orthomyxoviridae/metabolismo , Alveolos Pulmonares/inmunología , Alveolos Pulmonares/metabolismo , Receptores de Hidrocarburo de Aril/metabolismoRESUMEN
BACKGROUND: Elicitation of allergic contact dermatitis (ACD), an inflammatory type 4 hypersensitivity disease, induces skin infiltration by polyclonal effector CD8 αß T cells and precursors of tissue-resident memory T (TRM) cells. Because TRM have long-term potential to contribute to body-surface immunoprotection and immunopathology, their local regulation needs a fuller understanding. OBJECTIVE: We sought to investigate how TRM-cell maturation might be influenced by innate-like T cells pre-existing within many epithelia. METHODS: This study examined CD8+ TRM-cell maturation following hapten-induced ACD in wild-type mice and in strains harboring altered compartments of dendritic intraepidermal γδ T cells (DETCs), a prototypic tissue-intrinsic, innate-like T-cell compartment that reportedly regulates ACD, but by no elucidated mechanism. RESULTS: In addition to eliciting CD8 TRM, ACD induced DETC activation and an intimate coregulatory association of the 2 cell types. This depended on DETC sensing IFN-γ produced by CD8 cells and involved programmed death-ligand 1 (PD-L1). Thus, in mice lacking DETC or lacking IFN-γ receptor solely on γδ cells, ACD-elicited CD8 T cells showed enhanced proliferative and effector potentials and reduced motility, collectively associated with exaggerated ACD pathology. Comparable dysregulation was elicited by PD-L1 blockade in vitro, and IFN-γ-regulated PD-L1 expression was a trait of human skin-homing and intraepithelial γδ T cells. CONCLUSIONS: The size and quality of the tissue-infiltrating CD8 T-cell response during ACD can be profoundly regulated by local innate-like T cells responding to IFN-γ and involving PD-L1. Thus, interindividual and tissue-specific variations in tissue-intrinsic lymphocytes may influence responses to allergens and other challenges and may underpin inflammatory pathologies such as those repeatedly observed in γδ T-cell-deficient settings.
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Dermatitis Alérgica por Contacto , Interferón gamma , Animales , Humanos , Ratones , Antígeno B7-H1 , Linfocitos T CD8-positivos/patología , Dermatitis Alérgica por Contacto/patología , Piel/patologíaRESUMEN
The resolution of fluorescence microscopy images is limited by the physical properties of light. In the last decade, numerous super-resolution microscopy (SRM) approaches have been proposed to deal with such hindrance. Here we present Mean-Shift Super Resolution (MSSR), a new SRM algorithm based on the Mean Shift theory, which extends spatial resolution of single fluorescence images beyond the diffraction limit of light. MSSR works on low and high fluorophore densities, is not limited by the architecture of the optical setup and is applicable to single images as well as temporal series. The theoretical limit of spatial resolution, based on optimized real-world imaging conditions and analysis of temporal image stacks, has been measured to be 40 nm. Furthermore, MSSR has denoising capabilities that outperform other SRM approaches. Along with its wide accessibility, MSSR is a powerful, flexible, and generic tool for multidimensional and live cell imaging applications.
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Algoritmos , Medicamentos Genéricos , Sistemas de Lectura , Microscopía Fluorescente , Colorantes FluorescentesRESUMEN
Due to the wave nature of light, optical microscopy has a lower-bound lateral resolution limit of approximately half of the wavelength of visible light, that is, within the range of 200 to 350 nm. Fluorescence fluctuation-based super-resolution microscopy (FF-SRM) is a term used to encompass a collection of image analysis techniques that rely on the statistical processing of temporal variations of the fluorescence signal. FF-SRM aims to reduce the uncertainty of the location of fluorophores within an image, often improving spatial resolution by several tens of nanometers. FF-SRM is suitable for live-cell imaging due to its compatibility with most fluorescent probes and relatively simple instrumental and experimental requirements, which are mostly camera-based epifluorescence instruments. Each FF-SRM approach has strengths and weaknesses, which depend directly on the underlying statistical principles through which enhanced spatial resolution is achieved. In this review, the basic concepts and principles behind a range of FF-SRM methods published to date are described. Their operational parameters are explained and guidance for their selection is provided.
Due to light's wave nature, an optical microscope's resolution range is 200 to 350 nanometers. Several techniques enhance resolution; this work encompasses several fluorescence fluctuation super-resolution (FF-SMR) methods capable of achieving nanoscopic scales. FF-SRM is known to be suitable for fixed or live-cell imaging and compatible with most conventional microscope setups found in a laboratory. However, each FF-SRM approach has its strengths and weaknesses, which depend directly on the underlying principles through which enhanced spatial resolution is achieved. Therefore, the basic concepts and principles behind diverse FF-SRM methods are revisited in this review. In addition, their operational parameters are explained, and guidance for their selection is provided for microscopists interested in FF-SRM.
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Colorantes Fluorescentes , Procesamiento de Imagen Asistido por Computador , Microscopía Fluorescente/métodosRESUMEN
Biomedical imaging includes the use of a variety of techniques to study organs and tissues. Some of the possible imaging modalities are more spread at clinical level (CT, MRI, PET), while others, such as light and electron microscopy are preferred in life sciences research. The choice of the imaging modalities can be based on the capability to study functional aspects of an organism, the delivered radiation dose to the patient, and the achievable resolution. In the last few decades, spectroscopists and imaging scientists have been interested in the use of terahertz (THz) frequencies (30 µm to 3 mm wavelength) due to the low photon energy associated (Eâ¼1 meV, not causing breaking of the molecular bonds but still interacting with some vibrational modes) and the high penetration depth that is achievable. THz has been already adopted in security, quality control and material sciences. However, the adoption of THz frequencies for biological and clinical imaging means to face, as a major limitation, the very scarce resolution associated with the use of such long wavelengths. To address this aspect and reconcile the benefit of minimal harmfulness for bioimaging with the achievable resolving power, many attempts have been made. This review summarises the state-of-the-art of THz imaging applications aimed at achieving super-resolution, describing how practical aspects of optics and quasi-optics may be treated to efficaciously implement the use of THz as a new low-dose and versatile modality in biomedical imaging and clinical research.
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Microscopía , Imágen por Terahertz , Humanos , Imágen por Terahertz/métodosRESUMEN
The study of cellular and developmental processes in physiologically relevant three-dimensional (3D) systems facilitates an understanding of mechanisms underlying cell fate, disease and injury. While cutting-edge microscopy technologies permit the routine acquisition of 3D datasets, there is currently a limited number of open-source software packages to analyse such images. Here, we describe General Image Analysis of Nuclei-based Images (GIANI; https://djpbarry.github.io/Giani), new software for the analysis of 3D images. The design primarily facilitates segmentation of nuclei and cells, followed by quantification of morphology and protein expression. GIANI enables routine and reproducible batch-processing of large numbers of images, and comes with scripting and command line tools. We demonstrate the utility of GIANI by quantifying cell morphology and protein expression in confocal images of mouse early embryos and by segmenting nuclei from light-sheet microscopy images of the flour beetle embryo. We also validate the performance of the software using simulated data. More generally, we anticipate that GIANI will be a useful tool for researchers in a variety of biomedical fields.
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Imagenología Tridimensional , Microscopía , Algoritmos , Animales , Núcleo Celular , Procesamiento de Imagen Asistido por Computador/métodos , Imagenología Tridimensional/métodos , Ratones , Programas InformáticosRESUMEN
Tissues contain diverse cell populations that, together, make up physiologically functional units. A remarkable example is the animal epidermis, where neuronal and non-neuronal cells intermingle to allow somatosensory perception. In the peripheral nervous system (PNS), the tight association between heterogenous cell types poses challenges when the structural and physiological contributions of neuronal and surrounding cells need to be dissected with suitable precision. When genetic tools for cell-specific, spatiotemporally controlled gene expression are not available, targeted cell ablation represents a considerable obstacle. Here, we describe an efficient method to overcome this limitation and demonstrate its application to the study of the differentiating Drosophila epidermis and PNS. This methodology relies on the use of near infrared (NIR) femtosecond (fs) laser pulses for ablation of the desired cells at the desired time. We show how to confine the photodamage to the targeted cell to induce its death, without harming neighbouring tissues or structures. We validated our approach in the Drosophila PNS by studying the responses of photo-ablated neurons, non-neuronal cells, and the surrounding epidermis. Diverse cellular behaviours including cell extrusion, cell rearrangements and cell shape changes can be monitored in vivo immediately after damage, as well as for several hours post-ablation with high optical resolution using confocal microscopy. This methodology provides a flexible tool to ablate individual cells with high precision and study morphological responses to cell loss in targeted areas or neighbouring structures. We anticipate that this protocol can be easily adapted to other model systems and tissues.
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Autophagy is a conserved intracellular degradation pathway exerting various cytoprotective and homeostatic functions by using de novo double-membrane vesicle (autophagosome) formation to target a wide range of cytoplasmic material for vacuolar/lysosomal degradation. The Atg1 kinase is one of its key regulators, coordinating a complex signaling program to orchestrate autophagosome formation. Combining in vitro reconstitution and cell-based approaches, we demonstrate that Atg1 is activated by lipidated Atg8 (Atg8-PE), stimulating substrate phosphorylation along the growing autophagosomal membrane. Atg1-dependent phosphorylation of Atg13 triggers Atg1 complex dissociation, enabling rapid turnover of Atg1 complex subunits at the pre-autophagosomal structure (PAS). Moreover, Atg1 recruitment by Atg8-PE self-regulates Atg8-PE levels in the growing autophagosomal membrane by phosphorylating and thus inhibiting the Atg8-specific E2 and E3. Our work uncovers the molecular basis for positive and negative feedback imposed by Atg1 and how opposing phosphorylation and dephosphorylation events underlie the spatiotemporal regulation of autophagy.
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Autofagosomas/enzimología , Proteínas Relacionadas con la Autofagia/metabolismo , Autofagia , Proteínas Quinasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Autofagosomas/genética , Familia de las Proteínas 8 Relacionadas con la Autofagia/genética , Familia de las Proteínas 8 Relacionadas con la Autofagia/metabolismo , Proteínas Relacionadas con la Autofagia/genética , Activación Enzimática , Regulación Enzimológica de la Expresión Génica , Regulación Fúngica de la Expresión Génica , Fosforilación , Proteínas Quinasas/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Transducción de Señal , Factores de TiempoRESUMEN
NEUBIAS, the European Network of Bioimage Analysts, was created in 2016 with the goal of improving the communication and the knowledge transfer among the various stakeholders involved in the acquisition, processing and analysis of biological image data, and to promote the establishment and recognition of the profession of Bioimage Analyst. One of the most successful initiatives of the NEUBIAS programme was its series of 15 training schools, which trained over 400 new Bioimage Analysts, coming from over 40 countries. Here we outline the rationale behind the innovative three-level program of the schools, the curriculum, the trainer recruitment and turnover strategy, the outcomes for the community and the career path of analysts, including some success stories. We discuss the future of the materials created during this programme and some of the new initiatives emanating from the community of NEUBIAS-trained analysts, such as the NEUBIAS Academy. Overall, we elaborate on how this training programme played a key role in collectively leveraging Bioimaging and Life Science research by bringing the latest innovations into structured, frequent and intensive training activities, and on why we believe this should become a model to further develop in Life Sciences.
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Disciplinas de las Ciencias Biológicas , Instituciones Académicas , CurriculumRESUMEN
Neutrophils are innate immune cells involved in the elimination of pathogens and can also induce adaptive immune responses. Nα and Nß neutrophils have been described with distinct in vitro capacity to generate antigen-specific CD8 T-cell responses. However, how these cell types exert their role in vivo and how manipulation of Nß/Nα ratio influences vaccine-mediated immune responses are not known. In this study, we find that these neutrophil subtypes show distinct migratory and motility patterns and different ability to interact with CD8 T cells in the spleen following vaccinia virus (VACV) infection. Moreover, after analysis of adhesion, inflammatory, and migration markers, we observe that Nß neutrophils overexpress the α4ß1 integrin compared to Nα. Finally, by inhibiting α4ß1 integrin, we increase the Nß/Nα ratio and enhance CD8 T-cell responses to HIV VACV-delivered antigens. These findings provide significant advancements in the comprehension of neutrophil-based control of adaptive immune system and their relevance in vaccine design.
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The mechanisms by which prostate cancer shifts from an indolent castration-sensitive phenotype to lethal castration-resistant prostate cancer (CRPC) are poorly understood. Identification of clinically relevant genetic alterations leading to CRPC may reveal potential vulnerabilities for cancer therapy. Here we find that CUB domain-containing protein 1 (CDCP1), a transmembrane protein that acts as a substrate for SRC family kinases (SFKs), is overexpressed in a subset of CRPC. Notably, CDCP1 cooperates with the loss of the tumor suppressor gene PTEN to promote the emergence of metastatic prostate cancer. Mechanistically, we find that androgens suppress CDCP1 expression and that androgen deprivation in combination with loss of PTEN promotes the upregulation of CDCP1 and the subsequent activation of the SRC/MAPK pathway. Moreover, we demonstrate that anti-CDCP1 immunoliposomes (anti-CDCP1 ILs) loaded with chemotherapy suppress prostate cancer growth when administered in combination with enzalutamide. Thus, our study identifies CDCP1 as a powerful driver of prostate cancer progression and uncovers different potential therapeutic strategies for the treatment of metastatic prostate tumors.
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Antígenos de Neoplasias/biosíntesis , Moléculas de Adhesión Celular/biosíntesis , Regulación Neoplásica de la Expresión Génica , Sistema de Señalización de MAP Quinasas , Neoplasias de la Próstata/metabolismo , Regulación hacia Arriba , Animales , Antígenos de Neoplasias/genética , Benzamidas , Moléculas de Adhesión Celular/genética , Línea Celular Tumoral , Drosophila melanogaster , Humanos , Liposomas , Masculino , Nitrilos , Fosfohidrolasa PTEN/biosíntesis , Fosfohidrolasa PTEN/genética , Feniltiohidantoína/análogos & derivados , Feniltiohidantoína/farmacología , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patologíaRESUMEN
Tumor-associated macrophages (TAMs) represent a major component of the tumor microenvironment supporting tumorigenesis. TAMs re-education has been proposed as a strategy to promote tumor inhibition. However, whether this approach may work in prostate cancer is unknown. Here we find that Pten-null prostate tumors are strongly infiltrated by TAMs expressing C-X-C chemokine receptor type 2 (CXCR2), and activation of this receptor through CXCL2 polarizes macrophages toward an anti-inflammatory phenotype. Notably, pharmacological blockade of CXCR2 receptor by a selective antagonist promoted the re-education of TAMs toward a pro-inflammatory phenotype. Strikingly, CXCR2 knockout monocytes infused in Ptenpc-/-; Trp53pc-/- mice differentiated in tumor necrosis factor alpha (TNF-α)-releasing pro-inflammatory macrophages, leading to senescence and tumor inhibition. Mechanistically, PTEN-deficient tumor cells are vulnerable to TNF-α-induced senescence, because of an increase of TNFR1. Our results identify TAMs as targets in prostate cancer and describe a therapeutic strategy based on CXCR2 blockade to harness anti-tumorigenic potential of macrophages against this disease.
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Senescencia Celular , Macrófagos/patología , Neoplasias de la Próstata/patología , Receptores de Interleucina-8B/antagonistas & inhibidores , Animales , Carcinogénesis/metabolismo , Carcinogénesis/patología , Puntos de Control del Ciclo Celular , Línea Celular Tumoral , Polaridad Celular , Quimiocina CXCL2/administración & dosificación , Quimiocina CXCL2/farmacología , Humanos , Inflamación/patología , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Estadificación de Neoplasias , Pruebas de Neutralización , Fosfohidrolasa PTEN/metabolismo , Receptores de Interleucina-8B/metabolismo , Transducción de Señal/efectos de los fármacos , Factor de Necrosis Tumoral alfa/metabolismo , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
The early phase of influenza infection occurs in the upper respiratory tract and the trachea, but little is known about the initial events of virus recognition and control of viral dissemination by the immune system. Here, we report that inflammatory dendritic cells (IDCs) are recruited to the trachea shortly after influenza infection through type I interferon-mediated production of the chemokine CCL2. We further show that recruited IDCs express the C-type lectin receptor SIGN-R1, which mediates direct recognition of the virus by interacting with N-linked glycans present in glycoproteins of the virion envelope. Activation of IDCs via SIGN-R1 triggers the production of the chemokines CCL5, CXCL9 and CXCL10, which initiate the recruitment of protective natural killer (NK) cells in the infected trachea. In the absence of SIGN-R1, the recruitment and activation of NK cells is impaired, leading to uncontrolled viral proliferation. In sum, our results provide insight into the orchestration of the early cellular and molecular events involved in immune protection against influenza.
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Moléculas de Adhesión Celular/metabolismo , Células Dendríticas/inmunología , Virus de la Influenza A/inmunología , Lectinas Tipo C/metabolismo , Infecciones por Orthomyxoviridae/inmunología , Receptores de Superficie Celular/metabolismo , Animales , Quimiocinas/metabolismo , Modelos Animales de Enfermedad , Perros , Interferón Tipo I/metabolismo , Células Asesinas Naturales , Células de Riñón Canino Madin Darby , Ratones , Infecciones por Orthomyxoviridae/virología , Tráquea/inmunología , Tráquea/virologíaRESUMEN
Cancer stem cells (CSCs) contribute to disease progression and treatment failure in human cancers. The balance among self-renewal, differentiation, and senescence determines the expansion or progressive exhaustion of CSCs. Targeting these processes might lead to novel anticancer therapies. Here, we uncover a novel link between BRD4, mitochondrial dynamics, and self-renewal of prostate CSCs. Targeting BRD4 by genetic knockdown or chemical inhibitors blocked mitochondrial fission and caused CSC exhaustion and loss of tumorigenic capability. Depletion of CSCs occurred in multiple prostate cancer models, indicating a common vulnerability and dependency on mitochondrial dynamics. These effects depended on rewiring of the BRD4-driven transcription and repression of mitochondrial fission factor (Mff). Knockdown of Mff reproduced the effects of BRD4 inhibition, whereas ectopic Mff expression rescued prostate CSCs from exhaustion. This novel concept of targeting mitochondrial plasticity in CSCs through BRD4 inhibition provides a new paradigm for developing more effective treatment strategies for prostate cancer.
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Epigénesis Genética/genética , Mitocondrias/genética , Dinámicas Mitocondriales/genética , Células Madre Neoplásicas/metabolismo , Neoplasias de la Próstata/metabolismo , Animales , Ciclo Celular , Proliferación Celular , Senescencia Celular , Humanos , Masculino , Ratones , Ratones Desnudos , Mitocondrias/metabolismo , Mitocondrias/patología , Neoplasias de la Próstata/patología , Células Tumorales CultivadasRESUMEN
T cell dependent secretory IgA (SIgA) generated in the Peyer's patches (PPs) of the small intestine shapes a broadly diverse microbiota that is crucial for host physiology. The mutualistic co-evolution of host and microbes led to the relative tolerance of host's immune system towards commensal microorganisms. The ATP-gated ionotropic P2X7 receptor limits T follicular helper (Tfh) cells expansion and germinal center (GC) reaction in the PPs. Here we show that transient depletion of intestinal ATP can dramatically improve high-affinity IgA response against both live and inactivated oral vaccines. Ectopic expression of Shigella flexneri periplasmic ATP-diphosphohydrolase (apyrase) abolishes ATP release by bacteria and improves the specific IgA response against live oral vaccines. Antibody responses primed in the absence of intestinal extracellular ATP (eATP) also provide superior protection from enteropathogenic infection. Thus, modulation of eATP in the small intestine can affect high-affinity IgA response against gut colonizing bacteria.
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Adenosina Trifosfato/metabolismo , Gastroenteritis/inmunología , Microbioma Gastrointestinal/fisiología , Inmunoglobulina A Secretora/metabolismo , Infecciones por Salmonella/inmunología , Adenosina Trifosfato/inmunología , Administración Oral , Animales , Apirasa/inmunología , Apirasa/metabolismo , Proteínas Bacterianas/inmunología , Proteínas Bacterianas/metabolismo , Vacunas Bacterianas/administración & dosificación , Vacunas Bacterianas/inmunología , Modelos Animales de Enfermedad , Escherichia coli/inmunología , Escherichia coli/metabolismo , Femenino , Gastroenteritis/microbiología , Centro Germinal/inmunología , Centro Germinal/metabolismo , Humanos , Íleon/inmunología , Íleon/metabolismo , Íleon/microbiología , Inmunoglobulina A Secretora/inmunología , Mucosa Intestinal/inmunología , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiología , Ratones , Ratones Endogámicos C57BL , Ganglios Linfáticos Agregados/inmunología , Ganglios Linfáticos Agregados/metabolismo , Receptores Purinérgicos P2X7/inmunología , Receptores Purinérgicos P2X7/metabolismo , Infecciones por Salmonella/microbiología , Salmonella typhimurium/inmunología , Salmonella typhimurium/patogenicidad , Shigella flexneri/inmunología , Shigella flexneri/metabolismo , Linfocitos T Colaboradores-Inductores/inmunología , Linfocitos T Colaboradores-Inductores/metabolismoRESUMEN
Antibodies to the prion protein, PrP, represent a promising therapeutic approach against prion diseases but the neurotoxicity of certain anti-PrP antibodies has caused concern. Here we describe scPOM-bi, a bispecific antibody designed to function as a molecular prion tweezer. scPOM-bi combines the complementarity-determining regions of the neurotoxic antibody POM1 and the neuroprotective POM2, which bind the globular domain (GD) and flexible tail (FT) respectively. We found that scPOM-bi confers protection to prion-infected organotypic cerebellar slices even when prion pathology is already conspicuous. Moreover, scPOM-bi prevents the formation of soluble oligomers that correlate with neurotoxic PrP species. Simultaneous targeting of both GD and FT was more effective than concomitant treatment with the individual molecules or targeting the tail alone, possibly by preventing the GD from entering a toxic-prone state. We conclude that simultaneous binding of the GD and flexible tail of PrP results in strong protection from prion neurotoxicity and may represent a promising strategy for anti-prion immunotherapy.
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Anticuerpos Biespecíficos/farmacología , Cerebelo/inmunología , Inmunoterapia , Enfermedades por Prión/terapia , Proteínas Priónicas/inmunología , Priones/toxicidad , Animales , Anticuerpos Biespecíficos/inmunología , Células Cultivadas , Regiones Determinantes de Complementariedad/inmunología , Ratones , Ratones Transgénicos , Enfermedades por Prión/inmunología , Priones/inmunologíaRESUMEN
In the HTML version of this article initially published, the name of author Diletta Di Mitri was miscoded in the XML such that Di was included as part of the given name instead of the family name. The error has been corrected in the HTML version of the article.
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Maintenance of cellular proteostasis relies on efficient clearance of defective gene products. For misfolded secretory proteins, this involves dislocation from the endoplasmic reticulum (ER) into the cytosol followed by proteasomal degradation. However, polypeptide aggregation prevents cytosolic dislocation and instead activates ill-defined lysosomal catabolic pathways. Here, we describe an ER-to-lysosome-associated degradation pathway (ERLAD) for proteasome-resistant polymers of alpha1-antitrypsin Z (ATZ). ERLAD involves the ER-chaperone calnexin (CNX) and the engagement of the LC3 lipidation machinery by the ER-resident ER-phagy receptor FAM134B, echoing the initiation of starvation-induced, receptor-mediated ER-phagy. However, in striking contrast to ER-phagy, ATZ polymer delivery from the ER lumen to LAMP1/RAB7-positive endolysosomes for clearance does not require ER capture within autophagosomes. Rather, it relies on vesicular transport where single-membrane, ER-derived, ATZ-containing vesicles release their luminal content within endolysosomes upon membrane:membrane fusion events mediated by the ER-resident SNARE STX17 and the endolysosomal SNARE VAMP8. These results may help explain the lack of benefits of pharmacologic macroautophagy enhancement that has been reported for some luminal aggregopathies.
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Retículo Endoplásmico/metabolismo , Endosomas/metabolismo , Lisosomas/genética , Proteolisis , alfa 1-Antitripsina/metabolismo , Animales , Transporte Biológico Activo/fisiología , Calnexina/genética , Calnexina/metabolismo , Retículo Endoplásmico/genética , Endosomas/genética , Células HEK293 , Humanos , Péptidos y Proteínas de Señalización Intracelular , Proteínas de Membrana de los Lisosomas/genética , Proteínas de Membrana de los Lisosomas/metabolismo , Lisosomas/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Complejo de la Endopetidasa Proteasomal/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas Qa-SNARE/genética , Proteínas Qa-SNARE/metabolismo , Proteínas R-SNARE/genética , Proteínas R-SNARE/metabolismo , alfa 1-Antitripsina/genética , Proteínas de Unión al GTP rab/genética , Proteínas de Unión al GTP rab/metabolismo , Proteínas de Unión a GTP rab7RESUMEN
The mechanisms by which mitochondrial metabolism supports cancer anabolism remain unclear. Here, we found that genetic and pharmacological inactivation of pyruvate dehydrogenase A1 (PDHA1), a subunit of the pyruvate dehydrogenase complex (PDC), inhibits prostate cancer development in mouse and human xenograft tumor models by affecting lipid biosynthesis. Mechanistically, we show that in prostate cancer, PDC localizes in both the mitochondria and the nucleus. Whereas nuclear PDC controls the expression of sterol regulatory element-binding transcription factor (SREBF)-target genes by mediating histone acetylation, mitochondrial PDC provides cytosolic citrate for lipid synthesis in a coordinated manner, thereby sustaining anabolism. Additionally, we found that PDHA1 and the PDC activator pyruvate dehydrogenase phosphatase 1 (PDP1) are frequently amplified and overexpressed at both the gene and protein levels in prostate tumors. Together, these findings demonstrate that both mitochondrial and nuclear PDC sustain prostate tumorigenesis by controlling lipid biosynthesis, thus suggesting this complex as a potential target for cancer therapy.