RESUMEN
BACKGROUND: Vascular diseases are often treated minimally invasively. The interventional material (stents, guidewires, etc.) used during such percutaneous interventions are visualized by some form of image guidance. Today, this image guidance is usually provided by 2D X-ray fluoroscopy, that is, a live 2D image. 3D X-ray fluoroscopy, that is, a live 3D image, could accelerate existing and enable new interventions. However, existing algorithms for the 3D reconstruction of interventional material require either too many X-ray projections and therefore dose, or are only capable of reconstructing single, curvilinear structures. PURPOSE: Using only two new X-ray projections per 3D reconstruction, we aim to reconstruct more complex arrangements of interventional material than was previously possible. METHODS: This is achieved by improving a previously presented deep learning-based reconstruction pipeline, which assumes that the X-ray images are acquired by a continuously rotating biplane system, in two ways: (a) separation of the reconstruction of different object types, (b) motion compensation using spatial transformer networks. RESULTS: Our pipeline achieves submillimeter accuracy on measured data of a stent and two guidewires inside an anthropomorphic phantom with respiratory motion. In an ablation study, we find that the aforementioned algorithmic changes improve our two figures of merit by 75 % (1.76 mm â 0.44 mm) and 59 % (1.15 mm â 0.47 mm) respectively. A comparison of our measured dose area product (DAP) rate to DAP rates of 2D fluoroscopy indicates a roughly similar dose burden. CONCLUSIONS: This dose efficiency combined with the ability to reconstruct complex arrangements of interventional material makes the presented algorithm a promising candidate to enable 3D fluoroscopy.
Asunto(s)
Imagenología Tridimensional , Stents , Imagenología Tridimensional/métodos , Rayos X , Fluoroscopía/métodos , Fantasmas de Imagen , AlgoritmosRESUMEN
Mycobacteria survive in macrophages despite triggering pattern recognition receptors and T cell-derived IFN-γ production. Mycobacterial cord factor trehalose-6,6-dimycolate (TDM) binds the C-type lectin receptor MINCLE and induces inflammatory gene expression. However, the impact of TDM on IFN-γ-induced macrophage activation is not known. In this study, we have investigated the cross-regulation of the mouse macrophage transcriptome by IFN-γ and by TDM or its synthetic analogue trehalose-6,6-dibehenate (TDB). As expected, IFN-γ induced genes involved in Ag presentation and antimicrobial defense. Transcriptional programs induced by TDM and TDB were highly similar but clearly distinct from the response to IFN-γ. The glycolipids enhanced expression of a subset of IFN-γ-induced genes associated with inflammation. In contrast, TDM/TDB exerted delayed inhibition of IFN-γ-induced genes, including pattern recognition receptors, MHC class II genes, and IFN-γ-induced GTPases, with antimicrobial function. TDM downregulated MHC class II cell surface expression and impaired T cell activation by peptide-pulsed macrophages. Inhibition of the IFN-γ-induced GTPase GBP1 occurred at the level of transcription by a partially MINCLE-dependent mechanism that may target IRF1 activity. Although activation of STAT1 was unaltered, deletion of Socs1 relieved inhibition of GBP1 expression by TDM. Nonnuclear Socs1 was sufficient for inhibition, suggesting a noncanonical, cytoplasmic mechanism. Taken together, unbiased analysis of transcriptional reprogramming revealed a significant degree of negative regulation of IFN-γ-induced Ag presentation and antimicrobial gene expression by the mycobacterial cord factor that may contribute to mycobacterial persistence.
Asunto(s)
Factores Cordón/metabolismo , Proteínas de Unión al GTP/metabolismo , Inflamación/microbiología , Lectinas Tipo C/metabolismo , Macrófagos/fisiología , Proteínas de la Membrana/metabolismo , Mycobacterium tuberculosis/fisiología , Tuberculosis/microbiología , Animales , Presentación de Antígeno , Células Cultivadas , Proteínas de Unión al GTP/genética , Perfilación de la Expresión Génica , Humanos , Inflamación/inmunología , Interferón gamma/metabolismo , Lectinas Tipo C/genética , Activación de Macrófagos , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína 1 Supresora de la Señalización de Citocinas/genética , Proteína 1 Supresora de la Señalización de Citocinas/metabolismo , Tuberculosis/inmunologíaRESUMEN
The co-expression of miRNAs and their target proteins was studied on tissue microarrays from different prostate cancer (PCa) patients. PCa of primary Gleason pattern 4 (GP4), lymph node metastases of GP4, distant metastases, and normal tissue from the transitional and peripheral zones were co-stained by fluorescent miRNA in situ hybridization (miRisH) and protein immunohistofluorescence (IHF). The miRNAs and corresponding target proteins include the pairs miR-145/ERG, miR-143/uPAR, and miR-375/SEC23A. The fluorescence-stained and scanned tissue microarrays (TMAs) were evaluated by experienced uropathologists. The pair miR-145/ERG showed an exclusive staining for miR-145 in the nuclei of stromal cells, both in tumor and normal tissue, and for ERG in the cytoplasm with/without co-expression in the nucleus of tumor cells. The pair miR-143/uPAR revealed a clear distinction between miR-143 in the nuclei of stromal cells and uPAR staining in the cytoplasm of tumor cells. Metastases (lymph node and distant) however, showed tumor cells with cytoplasmic staining for miR-143/uPAR. In normal tissues, beside the nuclei of the stroma cells, gland cells could also express miR-143 and uPAR in the cytoplasm. miR-375 showed particular staining in the nucleoli of GP4 and metastatic samples, suggesting that nucleoli play a special role in sequestering proteins and miRNAs. Combined miRisH/IHF allows for the study of miRNA expression patterns and their target proteins at the single-cell level.
Asunto(s)
Técnica del Anticuerpo Fluorescente/métodos , Hibridación Fluorescente in Situ/métodos , MicroARNs/análisis , Neoplasias de la Próstata/química , Análisis de Matrices Tisulares , Humanos , Masculino , MicroARNs/metabolismo , Neoplasias de la Próstata/metabolismoRESUMEN
Interferon-gamma (IFN-γ) is a pleiotropic cytokine that exerts important functions in inflammation, infectious diseases, and cancer. The large GTPase human guanylate-binding protein 1 (GBP-1) is among the most strongly IFN-γ-induced cellular proteins. Previously, it has been shown that GBP-1 mediates manifold cellular responses to IFN-γ including the inhibition of proliferation, spreading, migration, and invasion and through this exerts anti-tumorigenic activity. However, the mechanisms of GBP-1 anti-tumorigenic activities remain poorly understood. Here, we elucidated the molecular mechanism of the human GBP-1-mediated suppression of proliferation by demonstrating for the first time a cross-talk between the anti-tumorigenic IFN-γ and Hippo pathways. The α9-helix of GBP-1 was found to be sufficient to inhibit proliferation. Protein-binding and molecular modeling studies revealed that the α9-helix binds to the DNA-binding domain of the Hippo signaling transcription factor TEA domain protein (TEAD) mediated by the 376VDHLFQK382 sequence at the N-terminus of the GBP-1-α9-helix. Mutation of this sequence resulted in abrogation of both TEAD interaction and suppression of proliferation. Further on, the interaction caused inhibition of TEAD transcriptional activity associated with the down-regulation of TEAD-target genes. In agreement with these results, IFN-γ treatment of the cells also impaired TEAD activity, and this effect was abrogated by siRNA-mediated inhibition of GBP-1 expression. Altogether, this demonstrated that the α9-helix is the proliferation inhibitory domain of GBP-1, which acts independent of the GTPase activity through the inhibition of the Hippo transcription factor TEAD in mediating the anti-proliferative cell response to IFN-γ.
Asunto(s)
Proliferación Celular , Proteínas de Unión al GTP/metabolismo , Interferón gamma/metabolismo , Mutación Missense , Factores de Transcripción/metabolismo , Proteínas de Unión al GTP/genética , Células HeLa , Humanos , Interferón gamma/genética , Dominios Proteicos , Estructura Secundaria de Proteína , Factores de Transcripción/genéticaRESUMEN
Time-lapse imaging of cell colonies in microfluidic chambers provides time series of bioimages, i.e., biomovies. They show the behavior of cells over time under controlled conditions. One of the main remaining bottlenecks in this area of research is the analysis of experimental data and the extraction of cell growth characteristics, such as lineage information. The extraction of the cell line by human observers is time-consuming and error-prone. Previously proposed methods often fail because of their reliance on the accurate detection of a single cell, which is not possible for high density, high diversity of cell shapes and numbers, and high-resolution images with high noise. Our task is to characterize subpopulations in biomovies. In order to shift the analysis of the data from individual cell level to cellular groups with similar fluorescence or even subpopulations, we propose to represent the cells by two new abstractions: the particle and the patch. We use a three-step framework: preprocessing, particle tracking, and construction of the patch lineage. First, preprocessing improves the signal-to-noise ratio and spatially aligns the biomovie frames. Second, cell sampling is performed by assuming particles, which represent a part of a cell, cell or group of contiguous cells in space. Particle analysis includes the following: particle tracking, trajectory linking, filtering, and color information, respectively. Particle tracking consists of following the spatiotemporal position of a particle and gives rise to coherent particle trajectories over time. Typical tracking problems may occur (e.g., appearance or disappearance of cells, spurious artifacts). They are effectively processed using trajectory linking and filtering. Third, the construction of the patch lineage consists in joining particle trajectories that share common attributes (i.e., proximity and fluorescence intensity) and feature common ancestry. This step is based on patch finding, patching trajectory propagation, patch splitting, and patch merging. The main idea is to group together the trajectories of particles in order to gain spatial coherence. The final result of CYCASP is the complete graph of the patch lineage. Finally, the graph encodes the temporal and spatial coherence of the development of cellular colonies. We present results showing a computation time of less than 5 min for biomovies and simulated films. The method, presented here, allowed for the separation of colonies into subpopulations and allowed us to interpret the growth of colonies in a timely manner.
RESUMEN
Corynebacterium diphtheriae is the causative agent of diphtheria, a toxin mediated disease of upper respiratory tract, which can be fatal. As a member of the CMNR group, C. diphtheriae is closely related to members of the genera Mycobacterium, Nocardia and Rhodococcus. Almost all members of these genera comprise an outer membrane layer of mycolic acids, which is assumed to influence host-pathogen interactions. In this study, three different C. diphtheriae strains were investigated in respect to their interaction with phagocytic murine and human cells and the invertebrate infection model Caenorhabditis elegans. Our results indicate that C. diphtheriae is able to delay phagolysosome maturation after internalization in murine and human cell lines. This effect is independent of the presence of mycolic acids, as one of the strains lacked corynomycolates. In addition, analyses of NF-κB induction revealed a mycolate-independent mechanism and hint to detrimental effects of the different strains tested on the phagocytic cells. Bioinformatics analyses carried out to elucidate the reason for the lack of mycolates in one of the strains led to the identification of a new gene involved in mycomembrane formation in C. diphtheriae.
Asunto(s)
Corynebacterium diphtheriae/genética , Difteria/microbiología , Interacciones Huésped-Patógeno/genética , Macrófagos/microbiología , Animales , Caenorhabditis elegans/genética , Caenorhabditis elegans/microbiología , Línea Celular , Corynebacterium diphtheriae/metabolismo , Corynebacterium diphtheriae/patogenicidad , Difteria/genética , Difteria/patología , Humanos , Macrófagos/metabolismo , Macrófagos/patología , Ratones , Mycobacterium/genética , Ácidos Micólicos/metabolismo , FN-kappa B/genética , Nocardia/genética , Fagosomas/microbiología , Rhodococcus/genéticaRESUMEN
BACKGROUND: Manual assessment and evaluation of fluorescent micrograph cell experiments is time-consuming and tedious. Automated segmentation pipelines can ensure efficient and reproducible evaluation and analysis with constant high quality for all images of an experiment. Such cell segmentation approaches are usually validated and rated in comparison to manually annotated micrographs. Nevertheless, manual annotations are prone to errors and display inter- and intra-observer variability which influence the validation results of automated cell segmentation pipelines. RESULTS: We present a new approach to simulate fluorescent cell micrographs that provides an objective ground truth for the validation of cell segmentation methods. The cell simulation was evaluated twofold: (1) An expert observer study shows that the proposed approach generates realistic fluorescent cell micrograph simulations. (2) An automated segmentation pipeline on the simulated fluorescent cell micrographs reproduces segmentation performances of that pipeline on real fluorescent cell micrographs. CONCLUSION: The proposed simulation approach produces realistic fluorescent cell micrographs with corresponding ground truth. The simulated data is suited to evaluate image segmentation pipelines more efficiently and reproducibly than it is possible on manually annotated real micrographs.
Asunto(s)
Algoritmos , Microscopía Fluorescente , Animales , Linfocitos B/citología , Linfocitos B/metabolismo , Forma de la Célula , Procesamiento de Imagen Asistido por Computador , Macrófagos/citología , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Protoplastos/citología , Protoplastos/metabolismoRESUMEN
The delta-retrovirus Human T-cell leukemia virus type 1 (HTLV-1) preferentially infects CD4+ T-cells via cell-to-cell transmission. Viruses are transmitted by polarized budding and by transfer of viral biofilms at the virological synapse (VS). Formation of the VS requires the viral Tax protein and polarization of the host cytoskeleton, however, molecular mechanisms of HTLV-1 cell-to-cell transmission remain incompletely understood. Recently, we could show Tax-dependent upregulation of the actin-bundling protein Fascin (FSCN-1) in HTLV-1-infected T-cells. Here, we report that Fascin contributes to HTLV-1 transmission. Using single-cycle replication-dependent HTLV-1 reporter vectors, we found that repression of endogenous Fascin by short hairpin RNAs and by Fascin-specific nanobodies impaired gag p19 release and cell-to-cell transmission in 293T cells. In Jurkat T-cells, Tax-induced Fascin expression enhanced virus release and Fascin-dependently augmented cell-to-cell transmission to Raji/CD4+ B-cells. Repression of Fascin in HTLV-1-infected T-cells diminished virus release and gag p19 transfer to co-cultured T-cells. Spotting the mechanism, flow cytometry and automatic image analysis showed that Tax-induced T-cell conjugate formation occurred Fascin-independently. However, adhesion of HTLV-1-infected MT-2 cells in co-culture with Jurkat T-cells was reduced upon knockdown of Fascin, suggesting that Fascin contributes to dissemination of infected T-cells. Imaging of chronically infected MS-9 T-cells in co-culture with Jurkat T-cells revealed that Fascin's localization at tight cell-cell contacts is accompanied by gag polarization suggesting that Fascin directly affects the distribution of gag to budding sites, and therefore, indirectly viral transmission. In detail, we found gag clusters that are interspersed with Fascin clusters, suggesting that Fascin makes room for gag in viral biofilms. Moreover, we observed short, Fascin-containing membrane extensions surrounding gag clusters and clutching uninfected T-cells. Finally, we detected Fascin and gag in long-distance cellular protrusions. Taken together, we show for the first time that HTLV-1 usurps the host cell factor Fascin to foster virus release and cell-to-cell transmission.
Asunto(s)
Proteínas Portadoras/metabolismo , Productos del Gen tax/metabolismo , Infecciones por HTLV-I/transmisión , Proteínas de Microfilamentos/metabolismo , Liberación del Virus/fisiología , Técnicas de Cocultivo , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Técnicas de Silenciamiento del Gen , Células HEK293 , Virus Linfotrópico T Tipo 1 Humano , Humanos , Immunoblotting , Células Jurkat , Microscopía Confocal , Reacción en Cadena de la Polimerasa , TransfecciónRESUMEN
Corynebacterium ulcerans is an emerging pathogen transmitted by a zoonotic pathway with a very broad host spectrum to humans. Despite rising numbers of infections and potentially fatal outcomes, data on the molecular basis of pathogenicity are scarce. In this study, the interaction of 2 C. ulcerans isolates - one from an asymptomatic dog, one from a fatal case of human infection - with human macrophages was investigated. C. ulcerans strains were able to survive in macrophages for at least 20 hours. Uptake led to delay of phagolysosome maturation and detrimental effects on the macrophages as deduced from cytotoxicity measurements and FACS analyses. The data presented here indicate a high infectious potential of this emerging pathogen.
Asunto(s)
Infecciones por Corynebacterium/microbiología , Infecciones por Corynebacterium/veterinaria , Corynebacterium/patogenicidad , Enfermedades de los Perros/microbiología , Macrófagos/microbiología , Macrófagos/patología , Anciano de 80 o más Años , Animales , Línea Celular , Corynebacterium/inmunología , Corynebacterium/aislamiento & purificación , Citocinas/inmunología , Enfermedades de los Perros/patología , Perros , Femenino , Humanos , Macrófagos/citología , Macrófagos/metabolismo , Fagosomas/microbiologíaRESUMEN
To evaluate macrophage spreading in immunofluorescence images of macrophages for surface protein CD11b and nuclear counterstaining with DAPI, it is necessary to measure the size of the macrophages at different time points after stimulation. Manual evaluation of fluorescent micrographs is usually a time-consuming and error-prone task, with poor reproducibility. Automatic image analysis methods can be used to improve the results. The quality of the analysis with these methods mainly depends on the quality of the image segmentation. A segmentation and quantification scheme based on shading correction, k-means clustering, and fast marching level sets has been developed for the purpose. An initial application of this approach showed that separating touching and overlapping cells in particular suffers severely in the inevitably blurred conditions, leading to partly erroneous measurements of macrophage spreading. An alternative method of segmentation in fluorescent micrographs was therefore investigated and evaluated in this study. The proposed approach uses a methodology that separates foreground objects from background objects on the basis of Boykov's graph cuts. In this process, a rough estimation of background pixels is used for background seeds. To identify foreground seeds, a difference of Gaussian band pass filter based workflow is developed. Information on foreground and background seeds is then used for a gradient magnitude based graph cut resulting in a robust figure-ground separation method. In addition, a fast marching level set approach is used in the post-processing step, which makes it possible to split touching cells by incorporating information about the cell nuclei. An evaluation based on a total of 553 manually labeled macrophages depicted in 21 micrographs showed that the proposed method significantly improves segmentation and splitting performance for fluorescent micrographs of LPS-stimulated macrophages and reduces the rate of error in automated analysis of macrophage spreading in comparison with alternative methods.