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IR-780 is a fluorescent marker, photostable and non-toxic, and is widely used in tumor targeting; however, studies on the impact of IR-780 in animal models of B16-F10 melanoma are scarce in the literature. Therefore, this study aims to analyze behavior of this marker in melanoma cells using in vitro and in vivo analyses with fluorescence microscopy to conduct an analysis of cell culture, and an in vivo imaging system for an analysis of cell culture, tumor targeting on animals, and organ examination. In vitro analysis showed that B16-F10 cells at a concentration of 2 × 105 cells.plate-1 allowed a better visualization using 20 µM of IR-780. Furthermore, the location of IR-780 accumulation was confirmed by its fluorescence microscopy. Through in vivo studies, fluorescence was not observed in subcutaneous nodules, and it was found that animals that received intraperitoneal injection of B16-F10 cells presented ascites and did not absorb IR-780. Additionally, animals exhibiting lung metastasis showed fluorescence in ex vivo lung images. Therefore, use of the IR-780 marker for evaluating the progression of tumor growth did not demonstrate efficiency; however, it was effective in diagnosing pulmonary metastatic tumors. Although this marker presented limitations, results of evaluating pulmonary involvement through ex vivo fluorescence imaging were determined based on intensity of fluorescence.
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Neoplasias Pulmonares , Melanoma Experimental , Neoplasias Cutáneas , Animales , Ratones , Neoplasias Pulmonares/diagnóstico por imagen , Neoplasias Pulmonares/secundario , Melanoma Experimental/diagnóstico por imagen , Melanoma Experimental/patología , Pulmón/patología , Ratones Endogámicos C57BLRESUMEN
PURPOSE: Both cyclic pentapeptide c(RGDfK) and acridine orange (AO) exhibit antitumor effects and cell permeability. This study aimed to evaluate the nuclear targeting efficiency and safety of the nuclear targeting probe for bladder cancer (BCa) synthesized by c(RGDfK) and AO. METHODS: The nuclear targeting probe AO-(cRGDfK)2 was synthesized from AO hydrochloride, azided c(RGDfK), and a near-infrared skeleton synthesized via click chemistry reactions. The effect of the AO-(cRGDfK)2 probe on cell viability was assessed in BCa 5637 cells. The tumor cell targeting efficacy of the AO-(cRGDfK)2 probe was evaluated in BCa cells in vitro and in tumor-bearing mice in vivo. Nuclear-specific accumulation of fluorescence probe in BCa tumor cells was evaluated using laser scanning confocal microscopy (LSCM). Hematoxylin and eosin staining was performed to detect histopathological changes in the spleen, heart, liver, and kidney. RESULTS: The AO-(cRGDfK)2 probe did not cause a significant reduction in cell viability. LSCM analysis showed that AO-(cRGDfK)2 exhibited nuclear-specific ambulation in BCa cells and was not accumulated in 293T cells. Also, this probe efficiently targeted tumor cells in the serum and urine samples. In vivo imaging system of tumor-bearing mice showed that ~ 80% percent of fluorescence signal was accumulated in the tumor sites. The probe did not change histopathology in the heart, liver, spleen, and kidney in tumor-bearing mice after the 21-day treatment. CONCLUSIONS: The AO-(cRGDfK)2 probe exhibited nuclear-specific accumulation in BCa cells without cytotoxicity, which provides an innovative alternative to improve anticancer therapy for BCa.
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Naranja de Acridina , Neoplasias de la Vejiga Urinaria , Animales , Ratones , Colorantes Fluorescentes , Neoplasias de la Vejiga Urinaria/tratamiento farmacológico , Eosina Amarillenta-(YS) , Riñón , Línea Celular TumoralRESUMEN
Spleen is a key organ for immunologic surveillance, acting as a firewall for antigens and parasites that spread through the blood. However, how spleen leukocytes evolve across the developmental phase, and how they spatially organize and interact in vivo is still poorly understood. Using a novel combination of selected antibodies and fluorophores to image in vivo the spleen immune environment, we described for the first time the dynamics of immune development across postnatal period. We found that spleens from adults and infants had similar numbers and arrangement of lymphoid cells. In contrast, splenic immune environment in newborns is sharply different from adults in almost all parameters analysed. Using this in vivo approach, B cells were the most frequent subtype throughout the development. Also, we revealed how infections - using a model of malaria - can change the spleen immune profile in adults and infants, which could become the key to understanding different severity grades of infection. Our new imaging solutions can be extremely useful for different groups in all areas of biological investigation, paving a way for new intravital approaches and advances.
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Malaria , Bazo , Adulto , Humanos , Recién Nacido , Microscopía Intravital , Linfocitos , Linfocitos BRESUMEN
Background: Radical prostatectomy, a standard management approach for localized Prostate Cancer (PC), may cause a stress response associated with immune modulating effects. Regional anesthesia was hypothesized to reduce the immune effects of surgery by minimizing the neuroendocrine surgical stress response, thus mitigating tumor cells dissemination. Our primary objective was to investigate whether the use of spinal blocks attenuates PC tumor cells dissemination on an animal model. We also assessed the number of circulating NK cells and the amount of inflammatory and anti-inflammatory cytokines. Materials and methods: A subcutaneous tumor model, with PC-3M cell line transfected with a luciferase-producing gene (PC-3M-luc-C6) was used. After proper tumor establishment and before tumors became metastatic, animals were submitted to tumor excision surgeries under general or combined (general and spinal) anesthesia. A control group was only anesthetized with general anesthesia. Results: The subcutaneous tumor model with PC-3M-luc-C6 cells was effective in causing distant metastasis after 35 days. The number of circulating tumor cells increased in animals that underwent surgery under general anesthesia alone compared to the group submitted to combined anesthesia. Interleukin 6 levels were different in all groups, with increase in the general anesthesia group. Conclusion: Our results suggest that combination of spinal and general anesthesia may attenuate the suppression of innate tumor immunity and it might be related to a reduction in the neuroendocrine response to surgery. Institutional protocol number: Animal Ethics Committee 1332/2019.
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Aim: Investigate the heterogeneous tumor tissue organization and examine how this condition can interfere with the passive delivery of a lipid nanoemulsion in two breast cancer preclinical models (4T1 and Ehrlich). Materials & methods: The authors used in vivo image techniques to follow the nanoemulsion biodistribution and microtomography, as well as traditional histopathology and electron microscopy to evaluate the tumor structural characteristics. Results & conclusion: Lipid nanoemulsion was delivered to the tumor, vascular organization depends upon the subtumoral localization and this heterogeneous organization promotes a nanoemulsion biodistribution to the highly vascular peripherical region. Also, the results are presented with a comprehensive mathematical model, describing the differential biodistribution in two different breast cancer models, the 4T1 and Ehrlich models.
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Neoplasias de la Mama , Nanopartículas , Humanos , Femenino , Línea Celular Tumoral , Distribución Tisular , Nanopartículas/química , Lípidos , Neoplasias de la Mama/diagnóstico por imagen , Emulsiones/químicaRESUMEN
The abundance of neutrophils in human circulation, their fast mobilization from blood to tissues, along with their alleged short life-span led to the image of neutrophils as a homogeneous cell type designed to fight infections and die in the process. Additionally, their granule content and capacity to produce molecules with considerable cytotoxic potential, lead to the general belief that neutrophil activation inexorably results in side effect of extensive tissue injury. Neutrophil activation in fact causes tissue injury as an adverse effect, but it seems that this is restricted to particular pathological situations and more of an "exception to the rule". Here we review evidences arising especially from intravital microscopy studies that demonstrate neutrophils as cells endowed with sophisticated mechanisms and able to engage in complex interactions as to minimize damage and optimize their effector functions. Moreover, neutrophil infiltration may even contribute to tissue healing and repair which may altogether demand a reexamination of current anti-inflammatory therapies that have neutrophil migration and activation as a target.
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Antiinflamatorios/farmacología , Inflamación/patología , Neutrófilos/metabolismo , Animales , Humanos , Inflamación/tratamiento farmacológico , Activación Neutrófila/fisiología , Infiltración Neutrófila/fisiologíaRESUMEN
Alzheimer's disease (AD), considered a common type of dementia, is mainly characterized by a progressive loss of memory and cognitive functions. Although its cause is multifactorial, it has been associated with the accumulation of toxic aggregates of the amyloid-ß peptide (Aß) and neurofibrillary tangles (NFTs) of tau protein. At present, the development of highly sensitive, high cost-effective, and non-invasive diagnostic tools for AD remains a challenge. In the last decades, nanomaterials have emerged as an interesting and useful tool in nanomedicine for diagnostics and therapy. In particular, plasmonic nanoparticles are well-known to display unique optical properties derived from their localized surface plasmon resonance (LSPR), allowing their use as transducers in various sensing configurations and enhancing detection sensitivity. Herein, this review focuses on current advances in in vitro sensing techniques such as Surface-enhanced Raman scattering (SERS), Surface-enhanced fluorescence (SEF), colorimetric, and LSPR using plasmonic nanoparticles for improving the sensitivity in the detection of main biomarkers related to AD in body fluids. Additionally, we refer to the use of plasmonic nanoparticles for in vivo imaging studies in AD.
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Enfermedad de Alzheimer , Nanopartículas del Metal , Enfermedad de Alzheimer/diagnóstico , Péptidos beta-Amiloides , Humanos , Espectrometría Raman , Resonancia por Plasmón de SuperficieRESUMEN
BACKGROUND & AIMS: The precise determination of non-alcoholic fatty liver disease (NAFLD) onset is challenging. Thus, the initial hepatic responses to fat accumulation, which may be fundamental to our understanding of NAFLD evolution and clinical outcomes, are largely unknown. Herein, we chronologically mapped the immunologic and metabolic changes in the liver during the early stages of fatty liver disease in mice and compared this with human NAFLD samples. METHODS: Liver biopsies from patients with NAFLD (NAFLD activity score [NAS] 2-3) were collected for gene expression profiling. Mice received a high-fat diet for short periods to mimic initial steatosis and the hepatic immune response was investigated using a combination of confocal intravital imaging, gene expression, cell isolation, flow cytometry and bone marrow transplantation assays. RESULTS: We observed major immunologic changes in patients with NAS 2-3 and in mice in the initial stages of NAFLD. In mice, these changes significantly increased mortality rates upon drug-induced liver injury, as well as predisposing mice to bacterial infections. Moreover, deletion of Toll-like receptor 4 in liver cells dampened tolerogenesis, particularly in Kupffer cells, in the initial stages of dietary insult. CONCLUSION: The hepatic immune system acts as a sentinel for early and minor changes in hepatic lipid content, mounting a biphasic response upon dietary insult. Priming of liver immune cells by gut-derived Toll-like receptor 4 ligands plays an important role in liver tolerance in initial phases, but continuous exposure to insults may lead to damage and reduced ability to control infections. LAY SUMMARY: Fatty liver is a very common form of hepatic disease, leading to millions of cases of cirrhosis every year. Patients are often asymptomatic until becoming very sick. Therefore, it is important that we expand our knowledge of the early stages of disease pathogenesis, to enable early diagnosis. Herein, we show that even in the early stages of fatty liver disease, there are significant alterations in genes involved in the inflammatory response, suggesting that the hepatic immune system is disturbed even following minor and undetectable changes in liver fat content. This could have implications for the diagnosis and clinical management of fatty liver disease.
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Neutrophils were classically described as powerful effectors of acute inflammation, and their main purpose was assumed to be restricted to pathogen killing through production of oxidants. As consequence, neutrophils also may lead to significant collateral damage to the healthy tissues, and after performing these tasks, these leukocytes are supposed to die within tissues. However, there is a growing body of evidence showing that neutrophils also play a pivotal role in the resolution phases of inflammation, because they can modulate tissue environment due to secretion of different kind of cytokines. Drug-induced liver injury (DILI) is a worldwide concern being one of the most prevalent causes of liver transplantation, and is well established that there is an intense neutrophil recruitment into necrotic liver during DILI. However, information if such abundant granulocyte infiltration is also linked to the tissue repairing phase of hepatic injury is still largely elusive. Here, we investigated the dynamics of neutrophil trafficking within blood, bone marrow, and liver during hepatic inflammation, and how changes in their gene expression profile could drive the resolution events during acetaminophen (APAP)-induced liver injury. We found that neutrophils remained viable during longer periods following liver damage, because they avidly patrolled necrotic areas and up-regulated pro-resolutive genes, including Tgfb, Il1r2, and Fpr2. Adoptive transference of "resolutive neutrophils" harvested from livers at 72 h after injury to mice at the initial phases of injury (6 h after APAP) significantly rescued organ injury. Thus, we provide novel insights on the role of neutrophils not only in the injury amplification, but also in the resolution phases of inflammation.
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Enfermedad Hepática Inducida por Sustancias y Drogas/inmunología , Hígado/inmunología , Infiltración Neutrófila , Neutrófilos/inmunología , Acetaminofén/efectos adversos , Acetaminofén/farmacología , Animales , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/inmunología , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Femenino , Hígado/patología , Ratones , Neutrófilos/patología , Receptores de Formil Péptido/inmunología , Receptores Tipo II de Interleucina-1/inmunología , Factor de Crecimiento Transformador beta/inmunología , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/inmunologíaRESUMEN
Leukocytes are a large population of cells spread within most tissues in the body. These cells may be either sessile (called as resident cells) or circulating leukocytes, which travel long journeys inside the vessels during their lifespan. Although production and maturation of these leukocytes in adults primarily occur in the bone marrow, it is well known that this process-called hematopoiesis-started in the embryonic life in different sites, including the yolk sac, placenta, and the liver. In this review, we will discuss how the liver acts as a pivotal site for leukocyte maturation during the embryo phase, and also how the most frequent liver-resident immune cell populations-namely Kupffer cells, dendritic cells, and lymphocytes-play a vital role in both tolerance and inflammatory responses to antigens from food, microbiota, and pathogens.
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Leucocitos/citología , Hígado/citología , Animales , Linaje de la Célula , Hematopoyesis , Humanos , Fagocitos/citologíaRESUMEN
In vivo and micro chemical analytical methods have the potential to improve our understanding of plant metabolism and development. Benchtop microprobe X-ray fluorescence spectroscopy (µ-XRF) presents a huge potential for facing this challenge. Excitation beams of 30 µm and 1 mm in diameter were employed to address questions in seed technology, phytopathology, plant physiology, and bioremediation. Different elements were analyzed in several situations of agronomic interest: (i) Examples of µ-XRF yielding quantitative maps that reveal the spatial distribution of zinc in common beans (Phaseolus vulgaris) primed seeds. (ii) Chemical images daily recorded at a soybean leaf (Glycine max) infected by anthracnose showed that phosphorus, sulfur, and calcium trended to concentrate in the disease spot. (iii) In vivo measurements at the stem of P. vulgaris showed that under root exposure, manganese is absorbed and transported nearly 10-fold faster than iron. (iv) Quantitative maps showed that the lead distribution in a leaf of Eucalyptus hybrid was not homogenous, this element accumulated mainly in the leaf border and midrib, the lead hotspots reached up to 13,400 mg lead kg-1 fresh tissue weight. These case studies highlight the ability of µ-XRF in performing qualitative and quantitative elemental analysis of fresh and living plant tissues. Thus, it can probe dynamic biological phenomena non-destructively and in real time.
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BACKGROUND & AIMS: The liver is the main hematopoietic site in embryos, becoming a crucial organ in both immunity and metabolism in adults. However, how the liver adapts both the immune system and enzymatic profile to challenges in the postnatal period remains elusive. We aimed to identify the mechanisms underlying this adaptation. METHODS: We analyzed liver samples from mice on day 0 after birth until adulthood. Human biopsies from newborns and adults were also examined. Liver immune cells were phenotyped using mass cytometry (CyTOF) and expression of several genes belonging to immune and metabolic pathways were measured. Mortality rate, bacteremia and hepatic bacterial retention after E. coli challenge were analyzed using intravital and in vitro approaches. In a set of experiments, mice were prematurely weaned and the impact on gene expression of metabolic pathways was evaluated. RESULTS: Human and mouse newborns have a sharply different hepatic cellular composition and arrangement compared to adults. We also found that myeloid cells and immature B cells primarily compose the neonatal hepatic immune system. Although neonatal mice were more susceptible to infections, a rapid evolution to an efficient immune response was observed. Concomitantly, newborns displayed a reduction of several macronutrient metabolic functions and the normal expression level of enzymes belonging to lipid and carbohydrate metabolism was reached around the weaning period. Interestingly, early weaning profoundly disturbed the expression of several hepatic metabolic pathways, providing novel insights into how dietary schemes affect the metabolic maturation of the liver. CONCLUSION: In newborns, the immune and metabolic profiles of the liver are dramatically different to those of the adult liver, which can be explained by the differences in the liver cell repertoire and phenotype. Also, dietary and antigen cues may be crucial to guide liver development during the postnatal phase. LAY SUMMARY: Newborns face major challenges in the extra-uterine life. In fact, organs need to modify their cellular composition and gene expression profile in order to adapt to changes in both microbiota and diet throughout life. The liver is interposed between the gastrointestinal system and the systemic circulation, being the destination of all macronutrients and microbial products from the gut. Therefore, it is expected that delicately balanced mechanisms govern the transformation of a neonatal liver to a key organ in adults.
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Recién Nacido , Hígado/inmunología , Hígado/metabolismo , Adulto , Animales , Animales Recién Nacidos , Biopsia , Infecciones por Escherichia coli/inmunología , Femenino , Hepatocitos , Humanos , Metabolismo de los Lípidos , Hígado/citología , Metaboloma , Ratones , Ratones Endogámicos C57BL , Células Progenitoras Mieloides/inmunología , Células Progenitoras Mieloides/fisiología , Valor Nutritivo/fisiología , Fagocitos/inmunología , Células Precursoras de Linfocitos B/inmunología , DesteteRESUMEN
Hepatocytes may rupture after a drug overdose, and their intracellular contents act as damage-associated molecular patterns (DAMPs) that lead to additional leukocyte infiltration, amplifying the original injury. Necrosis-derived DNA can be recognized as a DAMP, activating liver non-parenchymal cells (NPCs). We hypothesized that NPCs react to DNA by releasing interferon (IFN)-1, which amplifies acetaminophen (APAP)-triggered liver necrosis. We orally overdosed different knockout mouse strains to investigate the pathways involved in DNA-mediated amplification of APAP-induced necrosis. Mice were imaged under intravital confocal microscopy to estimate injury progression, and hepatocytes and liver NPCs were differentially isolated for gene expression assays. Flow cytometry (FACS) using a fluorescent reporter mouse estimated the interferon-beta production by liver leukocytes under different injury conditions. We also treated mice with DNase to investigate the role of necrosis DNA signaling in IFN-1 production. Hepatocytes released a large amount of DNA after APAP overdose, which was not primarily sensed by these cells. However, liver NPCs promptly sensed such environmental disturbances and activated several DNA sensing pathways. Liver NPCs synthesized and released IFN-1, which was associated with concomitant hepatocyte necrosis. Ablation of IFN-1 recognition in interferon α/ß receptor (IFNAR-/-) mice delayed APAP-mediated liver necrosis and dampened IFN-1 sensing pathways. We demonstrated a novel loop involving DNA recognition by hepatic NPCs and additional IFN-1 mediated hepatocyte death.
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The cytotoxic synapse formed between cytotoxic T lymphocytes or natural killer cells expressing CD95L and target cells with CD95 on their surface is a key pathway for apoptosis induction by the immune system. Despite similarities with the immune synapse in antigen presenting cells, little is known about the role of the spatiotemporal organization of agonistic proteins/receptor interactions for CD95 signaling. Here, we have developed an artificial cytotoxic synapse to examine how mobility and geometry of an anti-CD95 agonistic antibody affect receptor aggregation and mobility, ie the first step of receptor activation. By measuring the distribution, diffusion coefficient, and fraction of immobile CD95 receptor in living cells, we show that at short times, the initial activation of CD95 occurs locally and is limited to the contact region of the cytotoxic synapse. This anisotropic activation of apoptotic signaling supports a role for confined interactions on the efficiency of signal transduction that may have implications for biomedical applications of extrinsic apoptosis induction.
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Sinapsis/metabolismo , Receptor fas/metabolismo , Línea Celular , Membrana Celular/metabolismo , Proteína Ligando Fas/metabolismo , Recuperación de Fluorescencia tras Fotoblanqueo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Membrana Dobles de Lípidos , Espectrometría de Fluorescencia , Linfocitos T Citotóxicos/metabolismo , Receptor fas/genética , Receptor fas/inmunologíaRESUMEN
The aim of this study was to characterize the in vivo volumetric distribution of three folate-based biosensors by different imaging modalities (X-ray, fluorescence, Cerenkov luminescence, and radioisotopic imaging) through the development of a tridimensional image reconstruction algorithm. The preclinical and multimodal Xtreme imaging system, with a Multimodal Animal Rotation System (MARS), was used to acquire bidimensional images, which were processed to obtain the tridimensional reconstruction. Images of mice at different times (biosensor distribution) were simultaneously obtained from the four imaging modalities. The filtered back projection and inverse Radon transformation were used as main image-processing techniques. The algorithm developed in Matlab was able to calculate the volumetric profiles of 99mTc-Folate-Bombesin (radioisotopic image), 177Lu-Folate-Bombesin (Cerenkov image), and FolateRSense™ 680 (fluorescence image) in tumors and kidneys of mice, and no significant differences were detected in the volumetric quantifications among measurement techniques. The imaging tridimensional reconstruction algorithm can be easily extrapolated to different 2D acquisition-type images. This characteristic flexibility of the algorithm developed in this study is a remarkable advantage in comparison to similar reconstruction methods.
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Técnicas Biosensibles/métodos , Ácido Fólico/química , Imagenología Tridimensional , Imagen Molecular , Neoplasias/diagnóstico por imagen , Carga Tumoral , Administración Intravenosa , Algoritmos , Animales , Calibración , Ácido Fólico/administración & dosificación , Ratones Desnudos , Distribución TisularRESUMEN
Intravital imaging has been increasingly employed in cell biology studies and it is becoming one of the most powerful tools for in vivo investigation. Although some protocols can be extremely complex, most intravital imaging procedures can be performed using basic surgery and animal maintenance techniques. More importantly, regular confocal microscopes - the same that are used for imaging immunofluorescence slides - can also acquire high quality intravital images and movies after minor adaptations. Here we propose minimal adaptations in stock microscopes that allow major improvements in different fields of scientific investigation.
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Investigación Biomédica/instrumentación , Biología Celular/instrumentación , Microscopía Intravital , Microscopía Confocal , Animales , Investigación Biomédica/tendencias , Biología Celular/tendencias , Humanos , Sistema Inmunológico/diagnóstico por imagen , Microscopía Intravital/instrumentación , Microscopía Intravital/tendencias , Ratones , Microscopía Confocal/instrumentación , Microscopía Confocal/tendencias , Microscopía por Video/instrumentación , Microscopía por Video/tendenciasRESUMEN
Eastern, Venezuelan and western equine encephalitis viruses (EEEV, VEEV, and WEEV) are mosquito-borne viruses that cause substantial disease in humans and other vertebrates. Vaccines are limited and current treatment options have not proven successful. In this report, we vaccinated outbred mice with lipid-antigen-nucleic acid-complexes (LANACs) containing VEEV E1+WEEV E1 antigen and characterized protective efficacy against lethal EEEV, VEEV, and WEEV challenge. Vaccination resulted in complete protection against EEEV, VEEV, and WEEV in CD-1 mice. Measurements of bioluminescence and plaque reduction neutralization tests (PRNTs) indicate that LANAC VEEV E1+WEEV E1 vaccination is sterilizing against VEEV and WEEV challenge; whereas immunity to EEEV is not sterilizing. Passive transfer of rabbit VEEV E1+WEEV E1 immune serum to naive mice extended the mean time to death (MTD) of EEEV challenged mice and provided significant protection from lethal VEEV and WEEV challenge.
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Alphavirus/inmunología , Antígenos Virales/inmunología , Reacciones Cruzadas/inmunología , Virus de la Encefalitis Equina Venezolana/inmunología , Virus de la Encefalitis Equina del Oeste/inmunología , Proteínas Virales/inmunología , Infecciones por Alphavirus/inmunología , Infecciones por Alphavirus/mortalidad , Infecciones por Alphavirus/prevención & control , Infecciones por Alphavirus/virología , Animales , Anticuerpos Antivirales/inmunología , Antígenos Virales/administración & dosificación , Antígenos Virales/genética , Línea Celular , Modelos Animales de Enfermedad , Virus de la Encefalitis Equina Venezolana/genética , Virus de la Encefalitis Equina Venezolana/patogenicidad , Virus de la Encefalitis Equina del Oeste/genética , Virus de la Encefalitis Equina del Oeste/patogenicidad , Femenino , Expresión Génica , Genes Reporteros , Inmunidad Humoral , Inmunización , Liposomas , Ratones , Ácidos Nucleicos , Homología de Secuencia , Proteínas Virales/administración & dosificación , Proteínas Virales/genética , Virulencia/genética , Replicación ViralRESUMEN
Intracellular trafficking and asymmetric localization of RNA molecules within cells are a prevalent process across phyla involved in developmental control and signaling and thus in the determination of cell fate. In addition to intracellular localization, plants support the trafficking of RNA molecules also between cells through plasmodesmata (PD), which has important roles in the cell-to-cell and systemic communication during plant growth and development. Viruses have developed strategies to exploit the underlying plant RNA transport mechanisms for the cell-to-cell and systemic dissemination of infection. In vivo RNA visualization methods have revolutionized the study of RNA dynamics in living cells. However, their application in plants is still in its infancy. To gain insights into the RNA transport mechanisms in plants, we study the localization and transport of Tobacco mosaic virus RNA using MS2 tagging. This technique involves the tagging of the RNA of interest with repeats of an RNA stem-loop (SL) that is derived from the origin of assembly of the bacteriophage MS2 and recruits the MS2 coat protein (MCP). Thus, expression of MCP fused to a fluorescent marker allows the specific visualization of the SL-carrying RNA. Here we describe a detailed protocol for Agrobacterium tumefaciens-mediated transient expression and in vivo visualization of MS2-tagged mRNAs in Nicotiana benthamiana leaves.
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Nicotiana/virología , Imagen Óptica/métodos , Hojas de la Planta/virología , ARN Viral/análisis , Virus del Mosaico del Tabaco/aislamiento & purificación , Agrobacterium tumefaciens/genética , Proteínas de la Cápside/genética , Expresión Génica , Levivirus/genética , Microscopía Fluorescente/métodos , Hojas de la Planta/genética , Plantas Modificadas Genéticamente/genética , Transporte de ARN , ARN Viral/genética , ARN Viral/metabolismo , Nicotiana/genética , Virus del Mosaico del Tabaco/genética , Virus del Mosaico del Tabaco/metabolismoRESUMEN
Leishmania (L.) amazonensis [L. (L.) amazonensis] is widely distributed in Brazil and its symptomatic infections usually lead to few localized lesions and sometimes to diffuse cutaneous form, with nodules throughout the body, anergy to parasite antigens and poor therapeutic response. The variability of these manifestations draws attention to the need for studies on the pathophysiology of infection by this species. In this study, we analysed the course and immunological aspects of L. (L.) amazonensis infection in BALB/c and C57BL/6 strains, both susceptible, but displaying different clinical courses, and athymic BALB/c nude, to illustrate the role of T cell dependent responses. We analysed footpad thickness and parasite burden by in vivo imaging. Furthermore, we evaluated the cellular profile and cytokine production in lymph nodes and the inflammatory infiltrates of lesions. Nude mice showed delayed lesion development and less inflammatory cells in lesions, but higher parasite burden than BALB/c and C57BL/6. BALB/c and C57BL/6 mice had similar parasite burdens, lesion sizes and infiltrates until 6 weeks after infection, and after that C57BL/6 mice controlled the infection. Small differences in parasite numbers were observed in C57BL/6 macrophages in vitro, indicating that in vivo milieu accounts for most differences in infection. We believe our results shed light on the role of host immune system in the course of L. (L.) amazonensis infection by comparing three mouse strains that differ in parasitaemia and inflammatory cells.
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Interacciones Huésped-Parásitos/inmunología , Leishmaniasis Cutánea/inmunología , Animales , Citocinas/inmunología , Leishmania/inmunología , Ganglios Linfáticos/citología , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Desnudos , Carga de Parásitos , Especificidad de la EspecieRESUMEN
The liver has come a long way since it was considered only a metabolic organ attached to the gastrointestinal tract. The simultaneous ascension of immunology and intravital microscopy evidenced the liver as a central axis in the immune system, controlling immune responses to local and systemic agents as well as disease tolerance. The multiple hepatic cell populations are organized in a vascular environment that promotes intimate cellular interactions, including initiation of innate and adaptive immune responses, rapid leukocyte recruitment, pathogen clearance and production of a variety of immune mediators. In this review, we focus on the advances in liver immunology supported by intravital microscopy in diseases such as isquemia/reperfusion, acute liver injury and infections.