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1.
J Cancer Res Ther ; 16(2): 276-279, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32474513

RESUMO

Purpose: To study the arterial distribution of embosphere microsphere (EM) and polyvinyl alcohol (PVA) particles in rabbit mesenteric artery using in vivo microscopy.To study the arterial distribution of embosphere microsphere (EM) and polyvinyl alcohol (PVA) particles in rabbit mesenteric artery using in vivo microscopy. Methods: Sixteen New Zealand rabbits were divided into four groups, namely large PVA (560-710 µm), small PVA (150-350 µm), large EM (500-700 µm), and small EM (100-300 µm). The mesenteric arteries of the experimental animals were embolized under fluoroscopic guidance and visualized using in vivo microscopy. The embolized vessel diameter and arterial distribution of embolic agents were compared. Results: The diameters of occluded vessels in large PVA, small PVA, large EM, and small EM groups were 430.60 ± 67.30, 200.95 ± 70.54, 387.79 ± 92.51, and 143.81 ± 39.65 µm, respectively. PVA occluded significantly larger vessels than EM when the particle size was similar (P < 0.001). The proportion of EM at the bifurcation of the artery was significantly higher than that of PVA particles (large PVA < large EM, χ2 = 4.325, P < 0.038; small PVA < small EM, χ2 = 6.68, P < 0.01). Conclusion: Both PVA and EM could occlude vessels smaller than the particle size, and EM resulted in deeper penetration. The location of embolic particles in the artery is mainly related to the shape of particles.


Assuntos
Angiografia/métodos , Embolização Terapêutica/métodos , Microscopia Intravital/métodos , Artérias Mesentéricas/metabolismo , Microesferas , Álcool de Polivinil/farmacocinética , Animais , Sistemas de Liberação de Medicamentos/métodos , Artérias Mesentéricas/diagnóstico por imagem , Modelos Animais , Tamanho da Partícula , Álcool de Polivinil/química , Coelhos
2.
J Vis Exp ; (158)2020 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-32364551

RESUMO

Intravital microscopy (IVM) is widely used to monitor physiological and pathophysiological processes within the leukocyte recruitment cascade in vivo. The current protocol represents a practical and reproducible method to visualize the leukocyte endothelium interaction leading to leukocyte recruitment in skeletal muscle derived tissue within the intact organism of the mouse. The model is applicable to all fields of research that focus on granulocyte activation and their role in disease. We provide a step by step protocol to guide through the method and to highlight potential pitfalls and technical difficulties. The protocol covers the following aspects: experimental settings and required material, anesthesia of the mouse, dissection of the cremaster muscle as well as tracheal and carotid cannulation, IVM recordings and offline analysis. Data formats like adherent leukocytes, rolling flux (RF) and rolling flux fraction (RFF) are explained in detail and appropriate applications are discussed. Representative results from dystrophin deficient mdx mice are provided in the results section. IVM is a powerful tool to assess leukocyte recruitment in an in vivo setting; however, delineating for example endothelial and leukocyte function may require a combination with ex vivo setups like flow chamber experiments. Furthermore, the genetic background of animals of interest may greatly influence baseline recruitment, requiring individual fine tuning of the protocol provided. Despite its limitations, IVM may serve as a platform to readily translate in vitro findings into a living vertebrate organism.


Assuntos
Músculos Abdominais/fisiologia , Adesão Celular , Endotélio/metabolismo , Microscopia Intravital/métodos , Migração e Rolagem de Leucócitos , Leucócitos/fisiologia , Músculos Abdominais/diagnóstico por imagem , Animais , Endotélio/citologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos mdx
3.
Science ; 368(6487): 181-186, 2020 04 10.
Artigo em Inglês | MEDLINE | ID: mdl-32273467

RESUMO

Embryonic development is a complex process that is unamenable to direct observation. In this study, we implanted a window to the mouse uterus to visualize the developing embryo from embryonic day 9.5 to birth. This removable intravital window allowed manipulation and high-resolution imaging. In live mouse embryos, we observed transient neurotransmission and early vascularization of neural crest cell (NCC)-derived perivascular cells in the brain, autophagy in the retina, viral gene delivery, and chemical diffusion through the placenta. We combined the imaging window with in utero electroporation to label and track cell division and movement within embryos and observed that clusters of mouse NCC-derived cells expanded in interspecies chimeras, whereas adjacent human donor NCC-derived cells shrank. This technique can be combined with various tissue manipulation and microscopy methods to study the processes of development at unprecedented spatiotemporal resolution.


Assuntos
Embrião de Mamíferos/citologia , Embrião de Mamíferos/fisiologia , Desenvolvimento Embrionário , Microscopia Intravital/métodos , Crista Neural , Animais , Encéfalo/embriologia , Encéfalo/fisiologia , Divisão Celular , Movimento Celular , Quimera/embriologia , Quimera/fisiologia , Eletroporação , Feminino , Técnicas de Transferência de Genes , Camundongos , Camundongos Transgênicos , Neovascularização Fisiológica , Crista Neural/irrigação sanguínea , Crista Neural/citologia , Crista Neural/embriologia , Placenta/fisiologia , Gravidez , Retina/embriologia , Retina/fisiologia , Transmissão Sináptica , Útero
5.
PLoS Negl Trop Dis ; 14(3): e0008007, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32196491

RESUMO

Investigations into intracellular replication and differentiation of Trypanosoma cruzi within the mammalian host have been restricted by limitations in our ability to detect parasitized cells throughout the course of infection. We have overcome this problem by generating genetically modified parasites that express a bioluminescent/fluorescent fusion protein. By combining in vivo imaging and confocal microscopy, this has enabled us to routinely visualise murine infections at the level of individual host cells. These studies reveal that intracellular parasite replication is an asynchronous process, irrespective of tissue location or disease stage. Furthermore, using TUNEL assays and EdU labelling, we demonstrate that within individual infected cells, replication of both mitochondrial (kDNA) and nuclear genomes is not co-ordinated within the parasite population, and that replicating amastigotes and non-replicating trypomastigotes can co-exist in the same cell. Finally, we report the presence of distinct non-canonical morphological forms of T. cruzi in the mammalian host. These appear to represent transitional forms in the amastigote to trypomastigote differentiation process. Therefore, the intracellular life-cycle of T. cruzi in vivo is more complex than previously realised, with potential implications for our understanding of disease pathogenesis, immune evasion and drug development. Dissecting the mechanisms involved will be an important experimental challenge.


Assuntos
Doença de Chagas/parasitologia , Replicação do DNA , Estágios do Ciclo de Vida , Trypanosoma cruzi/crescimento & desenvolvimento , Animais , Modelos Animais de Doenças , Feminino , Genes Reporter , Microscopia Intravital/métodos , Camundongos SCID , Microscopia Confocal/métodos , Coloração e Rotulagem/métodos , Trypanosoma cruzi/genética
6.
PLoS One ; 15(2): e0219886, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32023253

RESUMO

Förster Resonance Energy Transfer (FRET) has become an immensely powerful tool to profile intra- and inter-molecular interactions. Through fusion of genetically encoded fluorescent proteins (FPs) researchers have been able to detect protein oligomerization, receptor activation, and protein translocation among other biophysical phenomena. Recently, two bright monomeric red fluorescent proteins, mRuby3 and mScarlet-I, have been developed. These proteins offer much improved physical properties compared to previous generations of monomeric red FPs that should help facilitate more general adoption of Green/Red FRET. Here we assess the ability of these two proteins, along with mCherry, to act as a FRET acceptor for the bright, monomeric, green-yellow FP mNeonGreen using intensiometric FRET and 2-photon Fluorescent Lifetime Imaging Microscopy (FLIM) FRET techniques. We first determined that mNeonGreen was a stable donor for 2-photon FLIM experiments under a variety of imaging conditions. We then tested the red FP's ability to act as FRET acceptors using mNeonGreen-Red FP tandem construct. With these constructs we found that mScarlet-I and mCherry are able to efficiently FRET with mNeonGreen in spectroscopic and FLIM FRET. In contrast, mNeonGreen and mRuby3 FRET with a much lower efficiency than predicted in these same assays. We explore possible explanations for this poor performance and determine mRuby3's protein maturation properties are a major contributor. Overall, we find that mNeonGreen is an excellent FRET donor, and both mCherry and mScarlet-I, but not mRuby3, act as practical FRET acceptors, with the brighter mScarlet-I out performing mCherry in intensiometric studies, but mCherry out performing mScarlet-I in instances where consistent efficiency in a population is critical.


Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Proteínas de Fluorescência Verde/metabolismo , Proteínas Luminescentes/normas , Transferência Ressonante de Energia de Fluorescência/normas , Células HEK293 , Humanos , Microscopia Intravital/métodos , Microscopia de Fluorescência/métodos , Análise de Célula Única/métodos
7.
Transplantation ; 104(6): e144-e150, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32080160

RESUMO

BACKGROUND: Current histological methods cannot accurately determine the survival rate of human pancreatic islets following portal vein infusion. This is due, in part, to the low number of infused islets relative to the whole liver. In this study, we assessed the ability of confocal laser scanning microscopy (CLSM) to track human islets posttransplantation. METHODS: Immunodeficient mice were transplanted with human islets. Following engraftment, animals were euthanized, livers procured, and human islet ß cells immunofluorescently labeled with an insulin-specific antibody and evaluated by CLSM. A calibration curve comparing the area of insulin + hepatic islet ß cells to the number of human islets collected was developed. Levels of human C-peptide were measured in transplant recipients to determine islet function. RESULTS: The short-term survival rate of islet transplants was defined as y = 0.0422x + 2.7008, in which x is human islet number and y is liver islet ß cell area. Employing CLSM, human islets were detected in immunofluorescent labeled murine liver tissue sections posttransplantation. The ß cell-relative area of human islets in 500 islet equivalent (IEQ) specimens was 20.21 ± 1.16 mm and in 1000 IEQ specimens 39.4 ± 2.23 mm posttransplantation. Human islet posttransplant survival rates were 82.9 ± 5.50% (500 IEQ group) and 86.9 ± 5.28% (1000 IEQ group). CONCLUSIONS: These data indicate that CLSM can be employed to quantify and characterize pancreatic human islets after transplantation to murine livers.


Assuntos
Rejeição de Enxerto/diagnóstico , Microscopia Intravital/métodos , Transplante das Ilhotas Pancreáticas/efeitos adversos , Ilhotas Pancreáticas/diagnóstico por imagem , Microscopia Confocal/métodos , Animais , Peptídeo C/análise , Peptídeo C/metabolismo , Estudos de Viabilidade , Rejeição de Enxerto/etiologia , Sobrevivência de Enxerto , Humanos , Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Masculino , Camundongos , Modelos Animais , Transplante Heterólogo
8.
J Invest Dermatol ; 140(2): 257-268.e8, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31980058

RESUMO

Pigmentation of the skin and hair represents the result of melanin biosynthesis within melanosomes of epidermal melanocytes, followed by the transfer of mature melanin granules to adjacent keratinocytes within the basal layer of the epidermis. Natural variation in these processes produces the diversity of skin and hair color among human populations, and defects in these processes lead to diseases such as oculocutaneous albinism. While genetic regulators of pigmentation have been well studied in human and animal models, we are still learning much about the cell biological features that regulate melanogenesis, melanosome maturation, and melanosome motility in melanocytes, and have barely scratched the surface in our understanding of melanin transfer from melanocytes to keratinocytes. Herein, we describe cultured cell model systems and common assays that have been used by investigators to dissect these features and that will hopefully lead to additional advances in the future.


Assuntos
Técnicas de Cultura de Células , Melaninas/análise , Melanossomas/química , Transtornos da Pigmentação/patologia , Pigmentação da Pele/fisiologia , Animais , Técnicas de Cocultura , Humanos , Processamento de Imagem Assistida por Computador , Microscopia Intravital/métodos , Queratinócitos/metabolismo , Melaninas/metabolismo , Melanossomas/metabolismo , Melanossomas/ultraestrutura , Microscopia Eletrônica de Transmissão/métodos , Microscopia de Fluorescência/métodos , Projetos de Pesquisa , Espectrofotometria/métodos
9.
PLoS One ; 15(1): e0227286, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31910228

RESUMO

In order to unravel rapid mechano-chemical feedback mechanisms in sprouting angiogenesis, we combine selective plane illumination microscopy (SPIM) and tailored image registration algorithms - further referred to as SPIM-based displacement microscopy - with an in vitro model of angiogenesis. SPIM successfully tackles the problem of imaging large volumes while upholding the spatial resolution required for the analysis of matrix displacements at a subcellular level. Applied to in vitro angiogenic sprouts, this unique methodological combination relates subcellular activity - minute to second time scale growing and retracting of protrusions - of a multicellular systems to the surrounding matrix deformations with an exceptional temporal resolution of 1 minute for a stack with multiple sprouts simultaneously or every 4 seconds for a single sprout, which is 20 times faster than with a conventional confocal setup. Our study reveals collective but non-synchronised, non-continuous activity of adjacent sprouting cells along with correlations between matrix deformations and protrusion dynamics.


Assuntos
Imageamento Tridimensional/métodos , Microscopia Intravital/métodos , Neovascularização Fisiológica/fisiologia , Imagem com Lapso de Tempo , Algoritmos , Técnicas de Cultura de Células/métodos , Colágeno Tipo I , Marcadores Fiduciais , Células Endoteliais da Veia Umbilical Humana , Humanos , Hidrogéis , Microscopia de Fluorescência/métodos , Microesferas
10.
PLoS Biol ; 18(1): e3000567, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31986129

RESUMO

Cell- and tissue-level processes often occur across days or weeks, but few imaging methods can capture such long timescales. Here, we describe Bellymount, a simple, noninvasive method for longitudinal imaging of the Drosophila abdomen at subcellular resolution. Bellymounted animals remain live and intact, so the same individual can be imaged serially to yield vivid time series of multiday processes. This feature opens the door to longitudinal studies of Drosophila internal organs in their native context. Exploiting Bellymount's capabilities, we track intestinal stem cell lineages and gut microbial colonization in single animals, revealing spatiotemporal dynamics undetectable by previously available methods.


Assuntos
Anatomia Transversal/métodos , Drosophila/anatomia & histologia , Microbioma Gastrointestinal , Microscopia Intravital/métodos , Vísceras/anatomia & histologia , Fatores Etários , Animais , Drosophila/microbiologia , Intestinos/anatomia & histologia , Intestinos/diagnóstico por imagem , Imagem Óptica/métodos , Vísceras/diagnóstico por imagem
11.
Nat Commun ; 11(1): 395, 2020 01 20.
Artigo em Inglês | MEDLINE | ID: mdl-31959752

RESUMO

Active nerve cells release vasodilators that increase their energy supply by dilating local blood vessels, a mechanism termed neurovascular coupling and the basis of BOLD functional neuroimaging signals. Here, we reveal a mechanism for cerebral blood flow control, a precapillary sphincter at the transition between the penetrating arteriole and first order capillary, linking blood flow in capillaries to the arteriolar inflow. The sphincters are encircled by contractile mural cells, which are capable of bidirectional control of the length and width of the enclosed vessel segment. The hemodynamic consequence is that precapillary sphincters can generate the largest changes in the cerebrovascular flow resistance of all brain vessel segments, thereby controlling capillary flow while protecting the downstream capillary bed and brain tissue from adverse pressure fluctuations. Cortical spreading depolarization constricts sphincters and causes vascular trapping of blood cells. Thus, precapillary sphincters are bottlenecks for brain capillary blood flow.


Assuntos
Capilares/fisiologia , Córtex Cerebral/irrigação sanguínea , Circulação Cerebrovascular/fisiologia , Contração Muscular/fisiologia , Músculo Liso Vascular/fisiologia , Animais , Capilares/diagnóstico por imagem , Córtex Cerebral/diagnóstico por imagem , Depressão Alastrante da Atividade Elétrica Cortical/fisiologia , Feminino , Neuroimagem Funcional/métodos , Imageamento Tridimensional , Microscopia Intravital/instrumentação , Microscopia Intravital/métodos , Masculino , Camundongos , Microscopia Confocal/instrumentação , Microscopia Confocal/métodos , Microscopia de Fluorescência por Excitação Multifotônica/instrumentação , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Modelos Animais , Modelos Cardiovasculares , Músculo Liso Vascular/diagnóstico por imagem , Fluxo Sanguíneo Regional/fisiologia , Crânio/cirurgia , Trepanação
12.
Artigo em Inglês | MEDLINE | ID: mdl-31994896

RESUMO

Airway mucus obstruction is a hallmark of chronic lung diseases such as cystic fibrosis, asthma, and COPD, and the development of more effective mucus-mobilizing therapies remains an important unmet need for patients with these muco-obstructive lung diseases. However, methods for sensitive visualization and quantitative assessment of immediate effects of therapeutic interventions on mucus clearance in vivo are lacking. In this study, we determined whether newly developed high-speed microscopic optical coherence tomography (mOCT) is sensitive to detect and compare in vivo effects of inhaled isotonic saline, hypertonic saline, and bicarbonate on mucus mobilization and clearance in Scnn1b-transgenic mice with muco-obstructive lung disease. In vivo mOCT imaging showed that inhaled isotonic saline-induced rapid mobilization of mucus that was mainly transported as chunks from the lower airways of Scnn1b-transgenic mice. Hypertonic saline mobilized a significantly greater amount of mucus that showed a more uniform distribution compared with isotonic saline. The addition of bicarbonate-to-isotonic saline had no effect on mucus mobilization, but also led to a more uniform mucus layer compared with treatment with isotonic saline alone. mOCT can detect differences in response to mucus-mobilizing interventions in vivo, and may thus support the development of more effective therapies for patients with muco-obstructive lung diseases.


Assuntos
Modelos Animais de Doenças , Canais Epiteliais de Sódio/fisiologia , Microscopia Intravital/métodos , Pneumopatias Obstrutivas/diagnóstico por imagem , Depuração Mucociliar , Muco/diagnóstico por imagem , Tomografia de Coerência Óptica/métodos , Animais , Humanos , Pneumopatias Obstrutivas/patologia , Pneumopatias Obstrutivas/terapia , Camundongos , Camundongos Transgênicos , Muco/fisiologia
13.
Int J Cancer ; 146(8): 2209-2217, 2020 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-31661557

RESUMO

Pancreatic cancers, both adenocarcinomas and endocrine tumors are characterized by varying levels of aberrant angiogenesis and fibrotic microenvironment. The difficulty to deliver drugs and treat the disease has been attributed in part to the vascular architecture and tissue/ECM density. Here we present longitudinal three-dimensional intravital imaging of vascular and tumor microenvironment remodeling in spontaneous transgenic tumors (RIP1-Tag2 insulinomas) and orthotopically injected tumors (KPC adenocarcinomas). Analysis of the data acquired in insulinomas revealed major differences in tumor blood vessel branching, fraction volume, number of branch points segments, vessel straightness and length compared to the normal tissue. The aggressive adenocarcinoma presented widespread peritumoral vascular remodeling and heterogeneous vascular distribution. Longitudinal imaging was used to acquire sequential vascular remodeling data during tumor progression. This work demonstrates the potential for using a pancreatic intravital imaging window for direct visualization of the tumor heterogenic microenvironments during tumor progression.


Assuntos
Neoplasias Pancreáticas/irrigação sanguínea , Neoplasias Pancreáticas/diagnóstico por imagem , Animais , Carcinoma Ductal Pancreático/irrigação sanguínea , Carcinoma Ductal Pancreático/diagnóstico por imagem , Linhagem Celular Tumoral , Matriz Extracelular , Microscopia Intravital/métodos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neovascularização Patológica/diagnóstico por imagem , Pâncreas/irrigação sanguínea , Microambiente Tumoral
14.
Nat Commun ; 10(1): 5647, 2019 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-31827103

RESUMO

Intravital microscopy is a powerful technique to observe dynamic processes with single-cell resolution in live animals. No intravital window has been developed for imaging the colon due to its anatomic location and motility, although the colon is a key organ where the majority of microbiota reside and common diseases such as inflammatory bowel disease, functional gastrointestinal disorders, and colon cancer occur. Here we describe an intravital murine colonic window with a stabilizing ferromagnetic scaffold for chronic imaging, minimizing motion artifacts while maximizing long-term survival by preventing colonic obstruction. Using this setup, we image fluorescently-labeled stem cells, bacteria, and immune cells in live animal colons. Furthermore, we image nerve activity via calcium imaging in real time to demonstrate that electrical sacral nerve stimulation can activate colonic enteric neurons. The simple implantable apparatus enables visualization of live processes in the colon, which will open the window to a broad range of studies.


Assuntos
Colo/diagnóstico por imagem , Microscopia Intravital/métodos , Imagem Óptica/métodos , Animais , Movimento Celular , Colo/microbiologia , Corantes Fluorescentes/química , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco/química , Células-Tronco/citologia
15.
Nat Commun ; 10(1): 5753, 2019 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-31848345

RESUMO

The coordination of cell movements across spatio-temporal scales ensures precise positioning of organs during vertebrate gastrulation. Mechanisms governing such morphogenetic movements have been studied only within a local region, a single germlayer or in whole embryos without cell identity. Scale-bridging imaging and automated analysis of cell dynamics are needed for a deeper understanding of tissue formation during gastrulation. Here, we report pan-embryo analyses of formation and dynamics of all three germlayers simultaneously within a developing zebrafish embryo. We show that a distinct distribution of cells in each germlayer is established during early gastrulation via cell movement characteristics that are predominantly determined by their position in the embryo. The differences in initial germlayer distributions are subsequently amplified by a global movement, which organizes the organ precursors along the embryonic body axis, giving rise to the blueprint of organ formation. The tools and data are available as a resource for the community.


Assuntos
Movimento Celular/fisiologia , Embrião não Mamífero/embriologia , Gastrulação/fisiologia , Camadas Germinativas/embriologia , Imagem Multimodal/métodos , Peixe-Zebra/embriologia , Animais , Embrião não Mamífero/diagnóstico por imagem , Camadas Germinativas/diagnóstico por imagem , Imageamento Tridimensional/métodos , Microscopia Intravital/métodos , Análise de Célula Única/métodos , Imagem com Lapso de Tempo/métodos
16.
Nat Commun ; 10(1): 5744, 2019 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-31848350

RESUMO

During bacterial cell division, the tubulin-homolog FtsZ forms a ring-like structure at the center of the cell. This Z-ring not only organizes the division machinery, but treadmilling of FtsZ filaments was also found to play a key role in distributing proteins at the division site. What regulates the architecture, dynamics and stability of the Z-ring is currently unknown, but FtsZ-associated proteins are known to play an important role. Here, using an in vitro reconstitution approach, we studied how the well-conserved protein ZapA affects FtsZ treadmilling and filament organization into large-scale patterns. Using high-resolution fluorescence microscopy and quantitative image analysis, we found that ZapA cooperatively increases the spatial order of the filament network, but binds only transiently to FtsZ filaments and has no effect on filament length and treadmilling velocity. Together, our data provides a model for how FtsZ-associated proteins can increase the precision and stability of the bacterial cell division machinery in a switch-like manner.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Transporte/metabolismo , Citocinese/fisiologia , Proteínas do Citoesqueleto/metabolismo , Citoesqueleto/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/fisiologia , Escherichia coli/citologia , Processamento de Imagem Assistida por Computador , Microscopia Intravital/métodos , Microscopia de Fluorescência/métodos , Imagem Individual de Molécula
17.
Virol J ; 16(1): 159, 2019 12 19.
Artigo em Inglês | MEDLINE | ID: mdl-31856881

RESUMO

BACKGROUND: Live-cell imaging is a powerful tool for visualization of the spatio-temporal dynamics of moving signals in living cells. Although this technique can be utilized to visualize nucleocapsid transport in Marburg virus (MARV)- or Ebola virus-infected cells, the experiments require biosafety level-4 (BSL-4) laboratories, which are restricted to trained and authorized individuals. METHODS: To overcome this limitation, we developed a live-cell imaging system to visualize MARV nucleocapsid-like structures using fluorescence-conjugated viral proteins, which can be conducted outside BSL-4 laboratories. RESULTS: Our experiments revealed that nucleocapsid-like structures have similar transport characteristics to those of nucleocapsids observed in MARV-infected cells, both of which are mediated by actin polymerization. CONCLUSIONS: We developed a non-infectious live cell imaging system to visualize intracellular transport of MARV nucleocapsid-like structures. This system provides a safe platform to evaluate antiviral drugs that inhibit MARV nucleocapsid transport.


Assuntos
Transporte Biológico , Microscopia Intravital/métodos , Marburgvirus/crescimento & desenvolvimento , Microscopia de Fluorescência/métodos , Nucleocapsídeo/metabolismo , Linhagem Celular , Avaliação Pré-Clínica de Medicamentos/métodos , Hepatócitos/virologia , Humanos , Processamento de Imagem Assistida por Computador/métodos , Coloração e Rotulagem/métodos , Proteínas Virais/análise
18.
Nat Commun ; 10(1): 5725, 2019 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-31844058

RESUMO

Many traits vary among isogenic individuals in homogeneous environments. In microbes, plants and animals, variation in the protein chaperone system affects many such traits. In the animal model C. elegans, the expression level of hsp-16.2 chaperone biomarkers correlates with or predicts the penetrance of mutations and lifespan after heat shock. But the physiological mechanisms causing cells to express different amounts of the biomarker were unknown. Here, we used an in vivo microscopy approach to dissect different contributions to cell-to-cell variation in hsp-16.2 expression in the intestines of young adult animals, which generate the most lifespan predicting signal. While we detected both cell autonomous intrinsic noise and signaling noise, we found both contributions were relatively unimportant. The major contributor to cell-to-cell variation in biomarker expression was general differences in protein dosage. The hsp-16.2 biomarker reveals states of high or low effective dosage for many genes.


Assuntos
Proteínas de Caenorhabditis elegans/genética , Dosagem de Genes , Proteínas de Choque Térmico/genética , Longevidade/genética , Penetrância , Animais , Animais Geneticamente Modificados , Biomarcadores/metabolismo , Caenorhabditis elegans/fisiologia , Proteínas de Caenorhabditis elegans/metabolismo , Genes Reporter/genética , Proteínas de Choque Térmico/metabolismo , Microscopia Intravital/métodos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia de Fluorescência/métodos , Modelos Animais , Imagem Molecular , Transdução de Sinais/genética
19.
Nat Commun ; 10(1): 5056, 2019 11 07.
Artigo em Inglês | MEDLINE | ID: mdl-31699983

RESUMO

Macrophages are one of the most functionally-diverse cell types with roles in innate immunity, homeostasis and disease making them attractive targets for diagnostics and therapy. Photo- or optoacoustics could provide non-invasive, deep tissue imaging with high resolution and allow to visualize the spatiotemporal distribution of macrophages in vivo. However, present macrophage labels focus on synthetic nanomaterials, frequently limiting their ability to combine both host cell viability and functionality with strong signal generation. Here, we present a homogentisic acid-derived pigment (HDP) for biocompatible intracellular labeling of macrophages with strong optoacoustic contrast efficient enough to resolve single cells against a strong blood background. We study pigment formation during macrophage differentiation and activation, and utilize this labeling method to track migration of pro-inflammatory macrophages in vivo with whole-body imaging. We expand the sparse palette of macrophage labels for in vivo optoacoustic imaging and facilitate research on macrophage functionality and behavior.


Assuntos
Ácido Homogentísico/química , Microscopia Intravital/métodos , Ativação de Macrófagos , Macrófagos/citologia , Técnicas Fotoacústicas/métodos , Pigmentos Biológicos/química , Coloração e Rotulagem/métodos , Animais , Materiais Biocompatíveis , Diferenciação Celular , Citocinas/metabolismo , Ouro , Células HEK293 , Células HeLa , Humanos , L-Lactato Desidrogenase/metabolismo , Macrófagos/metabolismo , Melaninas , Camundongos , Nanopartículas , Nanotubos
20.
Proc Natl Acad Sci U S A ; 116(48): 24275-24284, 2019 11 26.
Artigo em Inglês | MEDLINE | ID: mdl-31712432

RESUMO

T cells expressing anti-CD19 chimeric antigen receptors (CARs) demonstrate impressive efficacy in the treatment of systemic B cell malignancies, including B cell lymphoma. However, their effect on primary central nervous system lymphoma (PCNSL) is unknown. Additionally, the detailed cellular dynamics of CAR T cells during their antitumor reaction remain unclear, including their intratumoral infiltration depth, mobility, and persistence. Studying these processes in detail requires repeated intravital imaging of precisely defined tumor regions during weeks of tumor growth and regression. Here, we have combined a model of PCNSL with in vivo intracerebral 2-photon microscopy. Thereby, we were able to visualize intracranial PCNSL growth and therapeutic effects of CAR T cells longitudinally in the same animal over several weeks. Intravenous (i.v.) injection resulted in poor tumor infiltration of anti-CD19 CAR T cells and could not sufficiently control tumor growth. After intracerebral injection, however, anti-CD19 CAR T cells invaded deeply into the solid tumor, reduced tumor growth, and induced regression of PCNSL, which was associated with long-term survival. Intracerebral anti-CD19 CAR T cells entered the circulation and infiltrated distant, nondraining lymph nodes more efficiently than mock CAR T cells. After complete regression of tumors, anti-CD19 CAR T cells remained detectable intracranially and intravascularly for up to 159 d. Collectively, these results demonstrate the great potential of anti-CD19 CAR T cells for the treatment of PCNSL.


Assuntos
Neoplasias do Sistema Nervoso Central/terapia , Imunoterapia Adotiva/métodos , Microscopia Intravital/métodos , Linfoma/terapia , Linfócitos T/transplante , Animais , Antígenos CD19/análise , Antígenos CD19/imunologia , Antígenos CD19/metabolismo , Contagem de Células , Movimento Celular , Neoplasias do Sistema Nervoso Central/diagnóstico por imagem , Neoplasias do Sistema Nervoso Central/patologia , Citotoxicidade Imunológica , Fatores de Transcrição Forkhead/genética , Humanos , Injeções Intravenosas , Injeções Intraventriculares , Linfoma/diagnóstico por imagem , Linfoma/patologia , Masculino , Camundongos Mutantes , Neoplasias Experimentais/patologia , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/imunologia , Análise Espaço-Temporal , Linfócitos T/imunologia , Ensaios Antitumorais Modelo de Xenoenxerto
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