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1.
Photoacoustics ; 25: 100301, 2022 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-35036313

RESUMO

Test-samples are necessary for the development of emerging imaging approaches such as optoacoustics (OA); these can be used to benchmark new labeling agents and instrumentation, or to characterize image analysis algorithms or the inversion required to form the three-dimensional reconstructions. Alginate beads (AlBes) loaded with labeled mammalian or bacterial cells provide a method of creating defined structures of controllable size and photophysical characteristics and are well-suited for both in vitro and in vivo use. Here we describe a simple and rapid method for efficient and reproducible production of AlBes with specific characteristics and show three example applications with multispectral OA tomography imaging. We show the advantage of AlBes for studying and eventually improving photo-switching OA imaging approaches. As highly defined, homogeneous, quasi point-like signal sources, AlBes might hold similar advantages for studying other agents, light-fluence models, or the impact of detection geometries on correct image formation in the near future.

2.
Nat Biotechnol ; 40(4): 598-605, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-34845372

RESUMO

Reversibly photo-switchable proteins are essential for many super-resolution fluorescence microscopic and optoacoustic imaging methods. However, they have yet to be used as sensors that measure the distribution of specific analytes at the nanoscale or in the tissues of live animals. Here we constructed the prototype of a photo-switchable Ca2+ sensor based on GCaMP5G that can be switched with 405/488-nm light and describe its molecular mechanisms at the structural level, including the importance of the interaction of the core barrel structure of the fluorescent protein with the Ca2+ receptor moiety. We demonstrate super-resolution imaging of Ca2+ concentration in cultured cells and optoacoustic Ca2+ imaging in implanted tumor cells in mice under controlled Ca2+ conditions. Finally, we show the generalizability of the concept by constructing examples of photo-switching maltose and dopamine sensors based on periplasmatic binding protein and G-protein-coupled receptor-based sensors.


Assuntos
Técnicas Fotoacústicas , Animais , Linhagem Celular , Camundongos , Microscopia de Fluorescência/métodos , Técnicas Fotoacústicas/métodos
3.
Theranostics ; 11(16): 7813-7828, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34335966

RESUMO

Non-invasive monitoring of hemodynamic tumor responses to chemotherapy could provide unique insights into the development of therapeutic resistance and inform therapeutic decision-making in the clinic. Methods: Here, we examined the longitudinal and dynamic effects of the common chemotherapeutic drug Taxotere on breast tumor (KPL-4) blood volume and oxygen saturation using eigenspectra multispectral optoacoustic tomography (eMSOT) imaging over a period of 41 days. Tumor vascular function was assessed by dynamic oxygen-enhanced eMSOT (OE-eMSOT). The obtained in vivo optoacoustic data were thoroughly validated by ex vivo cryoimaging and immunohistochemical staining against markers of vascularity and hypoxia. Results: We provide the first preclinical evidence that prolonged treatment with Taxotere causes a significant drop in mean whole tumor oxygenation. Furthermore, application of OE-eMSOT showed a diminished vascular response in Taxotere-treated tumors and revealed the presence of static blood pools, indicating increased vascular permeability. Conclusion: Our work has important translational implications and supports the feasibility of eMSOT imaging for non-invasive assessment of tumor microenvironmental responses to chemotherapy.


Assuntos
Neoplasias da Mama/metabolismo , Hemodinâmica/fisiologia , Tomografia Óptica/métodos , Animais , Neoplasias da Mama/diagnóstico por imagem , Linhagem Celular Tumoral , Docetaxel/farmacologia , Feminino , Hemodinâmica/efeitos dos fármacos , Humanos , Hipóxia/metabolismo , Camundongos , Camundongos SCID , Oxigênio/metabolismo , Técnicas Fotoacústicas/métodos , Tomografia/métodos , Tomografia Computadorizada por Raios X/métodos , Microambiente Tumoral/fisiologia
4.
EMBO Mol Med ; 13(9): e13490, 2021 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-34411447

RESUMO

The increasing worldwide prevalence of obesity, fatty liver diseases and the emerging understanding of the important roles lipids play in various other diseases is generating significant interest in lipid research. Lipid visualization in particular can play a critical role in understanding functional relations in lipid metabolism. We investigated the potential of multispectral optoacoustic tomography (MSOT) as a novel modality to non-invasively visualize lipids in laboratory mice around the 930nm spectral range. Using an obesity-induced non-alcoholic fatty liver disease (NAFLD) mouse model, we examined whether MSOT could detect and differentiate different grades of hepatic steatosis and monitor the accumulation of lipids in the liver quantitatively over time, without the use of contrast agents, i.e. in label-free mode. Moreover, we demonstrate the efficacy of using the real-time clearance kinetics of indocyanine green (ICG) in the liver, monitored by MSOT, as a biomarker to evaluate the organ's function and assess the severity of NAFLD. This study establishes MSOT as an efficient imaging tool for lipid visualization in preclinical studies, particularly for the assessment of NAFLD.


Assuntos
Técnicas Fotoacústicas , Tomografia , Animais , Meios de Contraste , Verde de Indocianina , Camundongos , Tomografia Computadorizada por Raios X
5.
Photoacoustics ; 22: 100263, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33948433

RESUMO

Contrast enhancement in optoacoustic (photoacoustic) imaging can be achieved with agents that exhibit high absorption cross-sections, high photostability, low quantum yield, low toxicity, and preferential bio-distribution and clearance profiles. Based on advantageous photophysical properties of croconaine dyes, we explored croconaine-based nanoparticles (CR780RGD-NPs) as highly efficient contrast agents for targeted optoacoustic imaging of challenging preclinical tumor targets. Initial characterization of the CR780 dye was followed by modifications using polyethylene glycol and the cancer-targeting c(RGDyC) peptide, resulting in self-assembled ultrasmall particles with long circulation time and active tumor targeting. Preferential bio-distribution was demonstrated in orthotopic mouse brain tumor models by multispectral optoacoustic tomography (MSOT) imaging and histological analysis. Our findings showcase particle accumulation in brain tumors with sustainable strong optoacoustic signals and minimal toxic side effects. This work points to CR780RGD-NPs as a promising optoacoustic contrast agent for potential use in the diagnosis and image-guided resection of brain tumors.

6.
Nat Cell Biol ; 23(2): 184-197, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33462395

RESUMO

The transition zones of the squamous and columnar epithelia constitute hotspots for the emergence of cancer, often preceded by metaplasia, in which one epithelial type is replaced by another. It remains unclear how the epithelial spatial organization is maintained and how the transition zone niche is remodelled during metaplasia. Here we used single-cell RNA sequencing to characterize epithelial subpopulations and the underlying stromal compartment of endo- and ectocervix, encompassing the transition zone. Mouse lineage tracing, organoid culture and single-molecule RNA in situ hybridizations revealed that the two epithelia derive from separate cervix-resident lineage-specific stem cell populations regulated by opposing Wnt signals from the stroma. Using a mouse model of cervical metaplasia, we further show that the endocervical stroma undergoes remodelling and increases expression of the Wnt inhibitor Dickkopf-2 (DKK2), promoting the outgrowth of ectocervical stem cells. Our data indicate that homeostasis at the transition zone results from divergent stromal signals, driving the differential proliferation of resident epithelial lineages.


Assuntos
Colo do Útero/patologia , Epitélio/patologia , Homeostase , Via de Sinalização Wnt , Adenocarcinoma/genética , Adenocarcinoma/patologia , Animais , Biomarcadores Tumorais/metabolismo , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Diferenciação Celular , Linhagem da Célula , Microambiente Celular , Receptores ErbB/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Queratinas/metabolismo , Metaplasia , Camundongos Endogâmicos C57BL , Organoides/patologia , Receptores Notch/metabolismo , Células-Tronco/patologia , Células Estromais/patologia , Transcrição Genética , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/patologia
7.
Opt Express ; 28(24): 35427-35437, 2020 Nov 23.
Artigo em Inglês | MEDLINE | ID: mdl-33379657

RESUMO

Optical interrogation of tissues is broadly considered in biomedical applications. Nevertheless, light scattering by tissue limits the resolution and accuracy achieved when investigating sub-surface tissue features. Light carrying optical angular momentum or complex polarization profiles, offers different propagation characteristics through scattering media compared to light with unstructured beam profiles. Here we discuss the behaviour of structured light scattered by tissue-mimicking phantoms. We study the spatial and the polarization profile of the scattered modes as a function of a range of optical parameters of the phantoms, with varying scattering and absorption coefficients and of different lengths. These results show the non-trivial trade-off between the advantages of structured light profiles and mode broadening, stimulating further investigations in this direction.


Assuntos
Microscopia de Polarização/métodos , Imagens de Fantasmas , Espalhamento de Radiação , Biomimética , Luz , Modelos Biológicos
8.
Cancer Res ; 80(23): 5291-5304, 2020 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-32994204

RESUMO

Understanding temporal and spatial hemodynamic heterogeneity as a function of tumor growth or therapy affects the development of novel therapeutic strategies. In this study, we employed eigenspectra multispectral optoacoustic tomography (eMSOT) as a next-generation optoacoustic method to impart high accuracy in resolving tumor hemodynamics during bevacizumab therapy in two types of breast cancer xenografts (KPL-4 and MDA-MB-468). Patterns of tumor total hemoglobin concentration (THb) and oxygen saturation (sO2) were imaged in control and bevacizumab-treated tumors over the course of 58 days (KPL-4) and 16 days (MDA-MB-468), and the evolution of functional vasculature "normalization" was resolved macroscopically. An initial sharp drop in tumor sO2 and THb content shortly after the initiation of bevacizumab treatment was followed by a recovery in oxygenation levels. Rim-core subregion analysis revealed steep spatial oxygenation gradients in growing tumors that were reduced after bevacizumab treatment. Critically, eMSOT imaging findings were validated directly by histopathologic assessment of hypoxia (pimonidazole) and vascularity (CD31). These data demonstrate how eMSOT brings new abilities for accurate observation of entire tumor responses to challenges at spatial and temporal dimensions not available by other techniques today. SIGNIFICANCE: Accurate assessment of hypoxia and vascularization over space and time is critical for understanding tumor development and the role of spatial heterogeneity in tumor aggressiveness, metastasis, and response to treatment.


Assuntos
Bevacizumab/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Neovascularização Patológica/tratamento farmacológico , Inibidores da Angiogênese/farmacologia , Animais , Antineoplásicos Imunológicos/farmacologia , Neoplasias da Mama/irrigação sanguínea , Neoplasias da Mama/diagnóstico por imagem , Linhagem Celular Tumoral , Feminino , Humanos , Camundongos SCID , Neovascularização Patológica/diagnóstico por imagem , Oxigênio/metabolismo , Técnicas Fotoacústicas/métodos , Neoplasias de Mama Triplo Negativas/irrigação sanguínea , Neoplasias de Mama Triplo Negativas/diagnóstico por imagem , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/patologia , Ensaios Antitumorais Modelo de Xenoenxerto
9.
Sci Adv ; 6(24): eaaz6293, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32582850

RESUMO

We introduce two photochromic proteins for cell-specific in vivo optoacoustic (OA) imaging with signal unmixing in the temporal domain. We show highly sensitive, multiplexed visualization of T lymphocytes, bacteria, and tumors in the mouse body and brain. We developed machine learning-based software for commercial imaging systems for temporal unmixed OA imaging, enabling its routine use in life sciences.


Assuntos
Técnicas Fotoacústicas , Animais , Camundongos , Técnicas Fotoacústicas/métodos , Proteínas , Software
10.
IEEE Trans Biomed Eng ; 67(1): 185-192, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-30990172

RESUMO

OBJECTIVE: Fluorescence molecular imaging (FMI) has emerged as a promising tool for surgical guidance in oncology, with one of the few remaining challenges being the ability to offer quality control and data referencing. This paper investigates the use of a novel composite phantom to correct and benchmark FMI systems. METHODS: This paper extends on previous work by describing a phantom design that can provide a more complete assessment of FMI systems through quantification of dynamic range and determination of spatial illumination patterns for both reflectance and fluorescence imaging. Various performance metrics are combined into a robust and descriptive "system benchmarking score," enabling not only the comprehensive comparison of different systems, but also for the first time, correction of the acquired data. RESULTS: We show that systems developed for targeted fluorescence imaging can achieve benchmarking scores of up to 70%, while clinically available systems optimized for indocyanine green are limited to 50%, mostly due to greater leakage of ambient and excitation illumination and lower resolution. The image uniformity can also be approximated and employed for image flat-fielding, an important milestone toward data referencing. In addition, we demonstrate composite phantom use in assessing the performance of a surgical microscope and of a raster-scan imaging system. CONCLUSION: Our results suggest that the new phantom has the potential to support high-fidelity FMI through benchmarking and image correction. SIGNIFICANCE: Standardization of the FMI is a necessary process for establishing good imaging practices in clinical environments and for enabling high-fidelity imaging across patients and multi-center imaging studies.


Assuntos
Imagem Óptica , Imagens de Fantasmas/normas , Imagem Molecular/instrumentação , Imagem Molecular/normas , Imagem Óptica/instrumentação , Imagem Óptica/normas , Padrões de Referência
11.
Sci Rep ; 9(1): 18123, 2019 12 02.
Artigo em Inglês | MEDLINE | ID: mdl-31792293

RESUMO

Fluorescence imaging opens new possibilities for intraoperative guidance and early cancer detection, in particular when using agents that target specific disease features. Nevertheless, photon scattering in tissue degrades image quality and leads to ambiguity in fluorescence image interpretation and challenges clinical translation. We introduce the concept of capturing the spatially-dependent impulse response of an image and investigate Spatially Adaptive Impulse Response Correction (SAIRC), a method that is proposed for improving the accuracy and sensitivity achieved. Unlike classical methods that presume a homogeneous spatial distribution of optical properties in tissue, SAIRC explicitly measures the optical heterogeneity in tissues. This information allows, for the first time, the application of spatially-dependent deconvolution to correct the fluorescence images captured in relation to their modification by photon scatter. Using experimental measurements from phantoms and animals, we investigate the improvement in resolution and quantification over non-corrected images. We discuss how the proposed method is essential for maximizing the performance of fluorescence molecular imaging in the clinic.

12.
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
13.
Nat Commun ; 10(1): 1114, 2019 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-30846699

RESUMO

Advances in genetic engineering have enabled the use of bacterial outer membrane vesicles (OMVs) to deliver vaccines, drugs and immunotherapy agents, as a strategy to circumvent biocompatibility and large-scale production issues associated with synthetic nanomaterials. We investigate bioengineered OMVs for contrast enhancement in optoacoustic (photoacoustic) imaging. We produce OMVs encapsulating biopolymer-melanin (OMVMel) using a bacterial strain expressing a tyrosinase transgene. Our results show that upon near-infrared light irradiation, OMVMel generates strong optoacoustic signals appropriate for imaging applications. In addition, we show that OMVMel builds up intense heat from the absorbed laser energy and mediates photothermal effects both in vitro and in vivo. Using multispectral optoacoustic tomography, we noninvasively monitor the spatio-temporal, tumour-associated OMVMel distribution in vivo. This work points to the use of bioengineered vesicles as potent alternatives to synthetic particles more commonly employed for optoacoustic imaging, with the potential to enable both image enhancement and photothermal applications.


Assuntos
Nanopartículas , Técnicas Fotoacústicas/métodos , Animais , Proteínas da Membrana Bacteriana Externa/química , Bioengenharia , Biopolímeros/química , Feminino , Temperatura Alta/uso terapêutico , Neoplasias Mamárias Experimentais/diagnóstico por imagem , Neoplasias Mamárias Experimentais/terapia , Melaninas/química , Camundongos , Camundongos Nus , Nanopartículas/química , Nanotecnologia , Nanomedicina Teranóstica
14.
Nat Commun ; 10(1): 1191, 2019 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-30867430

RESUMO

Τhe morphology, physiology and immunology, of solid tumors exhibit spatial heterogeneity which complicates our understanding of cancer progression and therapy response. Understanding spatial heterogeneity necessitates high resolution in vivo imaging of anatomical and pathophysiological tumor information. We introduce Rhodobacter as bacterial reporter for multispectral optoacoustic (photoacoustic) tomography (MSOT). We show that endogenous bacteriochlorophyll a in Rhodobacter gives rise to strong optoacoustic signals >800 nm away from interfering endogenous absorbers. Importantly, our results suggest that changes in the spectral signature of Rhodobacter which depend on macrophage activity inside the tumor can be used to reveal heterogeneity of the tumor microenvironment. Employing non-invasive high resolution MSOT in longitudinal studies we show spatiotemporal changes of Rhodobacter spectral profiles in mice bearing 4T1 and CT26.WT tumor models. Accessibility of Rhodobacter to genetic modification and thus to sensory and therapeutic functions suggests potential for a theranostic platform organism.


Assuntos
Técnicas Biossensoriais/métodos , Macrófagos/imunologia , Neoplasias/diagnóstico por imagem , Técnicas Fotoacústicas/métodos , Rhodobacter/química , Nanomedicina Teranóstica/métodos , Animais , Bacterioclorofila A/química , Bacterioclorofila A/metabolismo , Linhagem Celular Tumoral/transplante , Modelos Animais de Doenças , Humanos , Estudos Longitudinais , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias/imunologia , Rhodobacter/metabolismo , Tomografia Computadorizada por Raios X/métodos , Microambiente Tumoral/imunologia
15.
J Biomed Opt ; 22(1): 16009, 2017 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-28301638

RESUMO

Fluorescence molecular imaging (FMI) has shown potential to detect and delineate cancer during surgery or diagnostic endoscopy. Recent progress on imaging systems has allowed sensitive detection of fluorescent agents even in video rate mode. However, lack of standardization in fluorescence imaging challenges the clinical application of FMI, since the use of different systems may lead to different results from a given study, even when using the same fluorescent agent. In this work, we investigate the use of a composite fluorescence phantom, employed as an FMI standard, to offer a comprehensive method for validation and standardization of the performance of different imaging systems. To exclude user interaction, all phantom features are automatically extracted from the acquired epi-illumination color and fluorescence images, using appropriately constructed templates. These features are then employed to characterize the performance and compare different cameras to each other. The proposed method could serve as a framework toward the calibration and benchmarking of FMI systems, to facilitate their clinical translation.


Assuntos
Benchmarking , Microscopia de Fluorescência/normas , Imagem Molecular/normas , Imagens de Fantasmas/normas , Calibragem , Microscopia de Fluorescência/instrumentação , Imagem Molecular/instrumentação , Imagem Óptica
16.
PLoS One ; 11(6): e0156898, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27294868

RESUMO

Norovirus infection is the main cause of epidemic non-bacterial gastroenteritis in humans. Although human norovirus (HuNoV) infection is self-limiting, it can persist for extended periods of time in immune deficient patients. Due to the lack of robust cell culture and small animal systems, little is known about HuNoV pathogenicity. However, murine norovirus (MNV) can be propagated in cell culture and is used as a model to study norovirus infection. Several MNV are known to persist in mice. In this study, we show that the MNV strain MNV-S99 persists in wild type inbred (C57BL/6J) mice over a period of at least 5 weeks post infection. Viral RNA was detectable in the jejunum, ileum, cecum, and colon, with the highest titers in the colon and cecum. To characterize the effect of MNV-S99 on the innate immune response, Stat1 phosphorylation and IFN-ß production were analyzed and compared to the non-persistent strain MNV-1.CW3. While MNV-S99 and MNV-1.CW3 showed comparable growth characteristics in vitro, Stat1 phosphorylation and IFN-ß release is strongly decreased after infection with MNV-S99 compared to MNV-1.CW3. In conclusion, our results show that unlike MNV-1.CW3, MNV-S99 establishes a persistent infection in mice, possibly due to interfering with the innate immune response.


Assuntos
Infecções por Caliciviridae/metabolismo , Interferon beta/metabolismo , Macrófagos/metabolismo , Norovirus/fisiologia , Fator de Transcrição STAT1/metabolismo , Animais , Infecções por Caliciviridae/imunologia , Infecções por Caliciviridae/virologia , Células Cultivadas , Farmacorresistência Viral Múltipla , Feminino , Gastroenterite/imunologia , Gastroenterite/metabolismo , Gastroenterite/virologia , Macrófagos/virologia , Camundongos , Camundongos Endogâmicos C57BL , Doenças dos Roedores/imunologia , Doenças dos Roedores/metabolismo , Doenças dos Roedores/virologia , Transdução de Sinais
17.
J Biomed Opt ; 21(9): 091309, 2016 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-27304578

RESUMO

Fluorescence imaging has been considered for over a half-century as a modality that could assist surgical guidance and visualization. The administration of fluorescent molecules with sensitivity to disease biomarkers and their imaging using a fluorescence camera can outline pathophysiological parameters of tissue invisible to the human eye during operation. The advent of fluorescent agents that target specific cellular responses and molecular pathways of disease has facilitated the intraoperative identification of cancer with improved sensitivity and specificity over nonspecific fluorescent dyes that only outline the vascular system and enhanced permeability effects. With these new abilities come unique requirements for developing phantoms to calibrate imaging systems and algorithms. We briefly review herein progress with fluorescence phantoms employed to validate fluorescence imaging systems and results. We identify current limitations and discuss the level of phantom complexity that may be required for developing a universal strategy for fluorescence imaging calibration. Finally, we present a phantom design that could be used as a tool for interlaboratory system performance evaluation.


Assuntos
Imagem Molecular/instrumentação , Imagem Óptica/instrumentação , Imagens de Fantasmas , Corantes Fluorescentes , Humanos , Modelos Biológicos , Poliuretanos
18.
Immunity ; 44(5): 1114-26, 2016 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-27192577

RESUMO

Regulatory T (Treg) cells expressing Foxp3 transcripton factor are essential for immune homeostasis. They arise in the thymus as a separate lineage from conventional CD4(+)Foxp3(-) T (Tconv) cells. Here, we show that the thymic development of Treg cells depends on the expression of their endogenous cognate self-antigen. The formation of these cells was impaired in mice lacking this self-antigen, while Tconv cell development was not negatively affected. Thymus-derived Treg cells were selected by self-antigens in a specific manner, while autoreactive Tconv cells were produced through degenerate recognition of distinct antigens. These distinct modes of development were associated with the expression of T cell receptor of higher functional avidity for self-antigen by Treg cells than Tconv cells, a difference subsequently essential for the control of autoimmunity. Our study documents how self-antigens define the repertoire of thymus-derived Treg cells to subsequently endow this cell type with the capacity to undermine autoimmune attack.


Assuntos
Antígeno CTLA-4/metabolismo , Encefalomielite Autoimune Experimental/imunologia , Esclerose Múltipla/imunologia , Glicoproteína Mielina-Oligodendrócito/metabolismo , Subpopulações de Linfócitos T/fisiologia , Linfócitos T Reguladores/fisiologia , Timo/imunologia , Animais , Autoantígenos/imunologia , Antígeno CTLA-4/genética , Células Cultivadas , Seleção Clonal Mediada por Antígeno , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Glicoproteína Mielina-Oligodendrócito/genética , Glicoproteína Mielina-Oligodendrócito/imunologia , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/metabolismo , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Especificidade do Receptor de Antígeno de Linfócitos T/genética
19.
Nat Commun ; 7: 11320, 2016 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-27177310

RESUMO

Chikungunya virus (CHIKV) is a globally spreading alphavirus against which there is no commercially available vaccine or therapy. Here we use a genome-wide siRNA screen to identify 156 proviral and 41 antiviral host factors affecting CHIKV replication. We analyse the cellular pathways in which human proviral genes are involved and identify druggable targets. Twenty-one small-molecule inhibitors, some of which are FDA approved, targeting six proviral factors or pathways, have high antiviral activity in vitro, with low toxicity. Three identified inhibitors have prophylactic antiviral effects in mouse models of chikungunya infection. Two of them, the calmodulin inhibitor pimozide and the fatty acid synthesis inhibitor TOFA, have a therapeutic effect in vivo when combined. These results demonstrate the value of loss-of-function screening and pathway analysis for the rational identification of small molecules with therapeutic potential and pave the way for the development of new, host-directed, antiviral agents.


Assuntos
Antivirais/farmacologia , Vírus Chikungunya/genética , Genoma Humano/genética , RNA Interferente Pequeno/genética , Replicação Viral/efeitos dos fármacos , Animais , Febre de Chikungunya/genética , Febre de Chikungunya/prevenção & controle , Febre de Chikungunya/virologia , Vírus Chikungunya/fisiologia , Furanos/farmacologia , Perfilação da Expressão Gênica/métodos , Células HEK293 , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Humanos , Camundongos , Pimozida/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Replicação Viral/genética
20.
Opt Lett ; 39(12): 3523-6, 2014 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-24978527

RESUMO

Multispectral optoacoustic tomography (MSOT) offers the potential to image in high-resolution cells tagged with optical labels. In contrast to single wavelength imaging, multispectral excitation and spectral unmixing can differentiate labeled moieties over tissue absorption in the absence of background measurements. This feature can enable longitudinal cellular biology studies well beyond the depths reached by optical microscopy. However, the relation between spectrally resolved fluorescently labeled cells and optoacoustic detection has not been systematically investigated. Herein, we measured titrations of fluorescently labeled cells and establish the optoacoustic signal generated by these cells as a function of cell number and across different cell types. We then assess the MSOT sensitivity to resolve cells implanted in animals.


Assuntos
Sistema Imunitário/citologia , Técnicas Fotoacústicas/métodos , Tomografia Óptica/métodos , Animais , Carbocianinas , Linhagem Celular , Corantes Fluorescentes , Humanos , Células Jurkat , Macrófagos/citologia , Macrófagos/imunologia , Macrófagos/transplante , Camundongos , Fenômenos Ópticos , Imagens de Fantasmas , Linfócitos T/citologia , Linfócitos T/imunologia
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