RESUMO
The environments that harbor hematopoietic stem and progenitor cells are critical to explore for a better understanding of hematopoiesis during health and disease. These compartments often are inaccessible for controlled and rapid experimentation, thus limiting studies to the evaluation of conventional cell culture and transgenic animal models. Here we describe the manufacture and image-guided monitoring of an engineered microenvironment with user-defined properties that recruits hematopoietic progenitors into the implant. Using intravital imaging and fluorescence molecular tomography, we show in real time that the cell homing and retention process is efficient and durable for short- and long-term engraftment studies. Our results indicate that bone marrow stromal cells, precoated on the implant, accelerate the formation of new sinusoidal blood vessels with vascular integrity at the microcapillary level that enhances the recruitment hematopoietic progenitor cells to the site. This implantable construct can serve as a tool enabling the study of hematopoiesis.
Assuntos
Células-Tronco Hematopoéticas/citologia , Neoplasias/patologia , Nicho de Células-Tronco , Alicerces Teciduais , Microambiente Tumoral , Animais , Matriz Extracelular , Humanos , Hidrogéis , Camundongos , Camundongos Endogâmicos NOD , Camundongos Nus , Microscopia Confocal , Tomografia/métodosRESUMO
BACKGROUND & AIMS: Bone marrow stromal cells (MSCs) are being evaluated as a cellular therapeutic for immune-mediated diseases. We investigated the effects of MSCs in mice with chemically induced colitis and determined the effects of CD11b(+) cells based on the hypothesis that MSCs increase numbers of regulatory T cells. METHODS: Colitis was induced in mice using trinitrobenzene sulfonic acid; symptoms were monitored as a function of MSC delivery. An immunomodulatory response was determined by measuring numbers of regulatory T cells in mesenteric lymph nodes. In vitro cocultures were used to assess the interaction of MSCs with regulatory T cells and CD11b(+) cells; findings were supported using near-infrared tracking of MSCs in vivo. We chemically and surgically depleted splenic CD11b(+) cells before colitis was induced with trinitrobenzene sulfonic acid to monitor the effects of MSCs. We adoptively transferred CD11b(+) cells that were cocultured with MSCs into mice with colitis. RESULTS: Intravenous grafts of MSCs prevented colitis and increased survival times of mice. Numbers of Foxp3(+) regulatory T cells increased in mesenteric lymph nodes in mice given MSCs. MSCs increased the numbers of Foxp3(+) splenocytes in a CD11b(+) cell-dependent manner. Transplanted MSCs colocalized near splenic CD11b(+) cells in vivo. Loss of CD11b(+) cells eliminated the therapeutic effect of MSCs. MSCs increased the anticolitis effects of CD11b(+) cells in mice. CONCLUSIONS: MSC transplants, delivered by specific parameters, reduce colitis in mice. Interactions between MSC and CD11b(+) regulatory T cells might be used to develop potency assays for MSCs, to identify nonresponders to MSC therapy, and to create new cell grafts that are composed of CD11b(+) cells preconditioned by MSCs.
Assuntos
Transplante de Medula Óssea , Antígeno CD11b/metabolismo , Colite/prevenção & controle , Colo/imunologia , Linfonodos/imunologia , Baço/imunologia , Células Estromais/transplante , Linfócitos T Reguladores/imunologia , Transferência Adotiva , Animais , Comunicação Celular , Rastreamento de Células , Técnicas de Cocultura , Colite/induzido quimicamente , Colite/imunologia , Colite/patologia , Colo/patologia , Modelos Animais de Doenças , Fatores de Transcrição Forkhead/metabolismo , Imunofenotipagem , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Células NIH 3T3 , Fenótipo , Fatores de Tempo , Ácido TrinitrobenzenossulfônicoRESUMO
We interrogate the ability of free-space fluorescence tomography to image small animals in vivo using charge-coupled device (CCD) camera measurements over 360-deg noncontact projections. We demonstrate the performance of normalized dual-wavelength measurements that are essential for in-vivo use, as they account for the heterogeneous distribution of photons in tissue. In-vivo imaging is then showcased on mouse lung and brain tumors cross-validated by x-ray microcomputed tomography and histology.
Assuntos
Neoplasias Encefálicas/patologia , Fluorescência , Processamento de Imagem Assistida por Computador/métodos , Neoplasias Pulmonares/patologia , Tomografia/métodos , Animais , Neoplasias Encefálicas/diagnóstico , Neoplasias Pulmonares/diagnóstico , Camundongos , Reprodutibilidade dos Testes , Microtomografia por Raio-XRESUMO
The availability of fluorescence standards is necessary in the development of systems and methods for fluorescence imaging. In this study, two approaches for developing diffuse fluorescence materials to be used as standards or phantoms in diffuse fluorescent tomography applications were investigated. Specifically, silicone rubber and polyester casting resin were used as base materials, and silicone pigments or TiO(2) / India Ink were added respectively to vary the optical properties. Characterization of the optical properties achieved was performed using time-resolved methods. Subsequently, different near-infrared fluorochromes were examined for imparting controlled and stable fluorescence properties. It was determined that hydrophobic fluorophores (IR 676 and IR 780 Iodide) suspended in dichloromethane and hydrophilic fluorophores (Cy5.5 and AF 750) suspended in methanol produced diffusive silicone and resin fluorescent materials, respectively. However only the hydrophobic fluorophores embedded within silicone resulted in the construction of a material with the characteristics of a standard, i.e. stability of fluorescence intensity with time and a linear dependence of normalized fluorescence intensity to fluorophore concentration.
RESUMO
Fluorescence imaging of tissues has gained significant attention in recent years due to the emergence of appropriate reporter technologies that enable noninvasive sensing of molecular function in vivo. Two major approaches have been used so far for fluorescence molecular imaging, i.e., epi-illumination (reflectance) imaging and fluorescence molecular tomography. Transillumination is an alternative approach that has been employed for imaging tissues in the past and could be similarly beneficial for fluorescence molecular imaging. We investigate data normalization schemes in reflectance and transillumination mode and experimentally demonstrate that normalized transillumination offers significant advantages over planar reflectance imaging and over nonnormalized methods. Our observations, based on phantoms and on postmortem and in vivo mouse measurements display image quality improvement, superior depth sensitivity, and improved imaging accuracy over the nonnormalized methods examined. Normalized planar imaging retains implementation simplicity and could be used to improve on standard fluorescence reflectance imaging and as a simplified alternative to the more integrated and accurate tomographic methods.
Assuntos
Algoritmos , Aumento da Imagem/métodos , Interpretação de Imagem Assistida por Computador/métodos , Neoplasias Mamárias Experimentais/patologia , Microscopia de Fluorescência/métodos , Imagem Corporal Total/métodos , Animais , Interpretação Estatística de Dados , Camundongos , Imagens de Fantasmas , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Observing drug responses in the tumor microenvironment in vivo can be technically challenging. As a result, cellular responses to molecularly targeted cancer drugs are often studied in cell culture, which does not accurately represent the behavior of cancer cells growing in vivo. Using high-resolution microscopy and fluorescently labeled genetic reporters for apoptosis, we developed an approach to visualize drug-induced cell death at single-cell resolution in vivo. Stable expression of the mitochondrial intermembrane protein IMS-RP was established in human breast and pancreatic cancer cells. Image analysis was then used to quantify release of IMS-RP into the cytoplasm upon apoptosis and irreversible mitochondrial permeabilization. Both breast and pancreatic cancer cells showed higher basal apoptotic rates in vivo than in culture. To study drug-induced apoptosis, we exposed tumor cells to navitoclax (ABT-263), an inhibitor of Bcl-2, Bcl-xL, and Bcl-w, both in vitro and in vivo. Although the tumors responded to Bcl-2 inhibition in vivo, inducing apoptosis in around 20% of cancer cells, the observed response was much higher in cell culture. Together, our findings show an imaging technique that can be used to directly visualize cell death within the tumor microenvironment in response to drug treatment.
Assuntos
Compostos de Anilina/farmacologia , Antineoplásicos/farmacologia , Apoptose , Neoplasias da Mama/fisiopatologia , Membranas Mitocondriais/fisiologia , Imagem Molecular/métodos , Neoplasias Pancreáticas/fisiopatologia , Sulfonamidas/farmacologia , Animais , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral , Feminino , Proteínas de Fluorescência Verde , Humanos , Processamento de Imagem Assistida por Computador , Camundongos , Camundongos Nus , Membranas Mitocondriais/efeitos dos fármacos , Proteínas Mitocondriais/metabolismo , Neoplasias Pancreáticas/tratamento farmacológico , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Proteínas Recombinantes de Fusão/metabolismo , Análise de Célula Única , Microambiente TumoralRESUMO
OBJECTIVE: To study the in vitro effects of poly(lactic-co-glycolic acid) (PLGA) nanoparticles loaded with the photosensitizer methylene blue (MB) and light against Enterococcus faecalis (ATCC 29212). MATERIALS AND METHODS: The uptake and distribution of nanoparticles in E. faecalis in suspension was investigated by transmission electron microscopy (TEM) after incubation with PLGA complexed with colloidal gold particles for 2.5, 5, and 10 minutes. E. faecalis species were sensitized in planktonic phase and in experimentally infected root canals of human extracted teeth with MB-loaded nanoparticles for 10 minutes followed by exposure to red light at 665 nm. RESULTS: The nanoparticles were found to be concentrated mainly on the cell walls of microorganisms at all three time points. The synergism of light and MB-loaded nanoparticles led to approximately 2 and 1 log10 reduction of colony-forming units (CFUs) in planktonic phase and root canals, respectively. In both cases, mean log10 CFU levels were significantly lower than controls and MB-loaded nanoparticles without light. CONCLUSION: The utilization of PLGA nanoparticles encapsulated with photoactive drugs may be a promising adjunct in antimicrobial endodontic treatment.
Assuntos
Anti-Infecciosos Locais/administração & dosagem , Ácido Láctico/administração & dosagem , Azul de Metileno/administração & dosagem , Nanopartículas/administração & dosagem , Fotoquimioterapia/métodos , Fármacos Fotossensibilizantes/administração & dosagem , Ácido Poliglicólico/administração & dosagem , Anti-Infecciosos Locais/química , Contagem de Colônia Microbiana , Cavidade Pulpar/microbiologia , Desinfecção/métodos , Portadores de Fármacos/administração & dosagem , Portadores de Fármacos/química , Enterococcus faecalis/efeitos dos fármacos , Enterococcus faecalis/efeitos da radiação , Enterococcus faecalis/ultraestrutura , Humanos , Ácido Láctico/química , Azul de Metileno/química , Nanopartículas/química , Nanopartículas/ultraestrutura , Fármacos Fotossensibilizantes/química , Ácido Poliglicólico/química , Copolímero de Ácido Poliláctico e Ácido PoliglicólicoRESUMO
We have found that broadband light (380 to 520 nm) rapidly and selectively kills oral black-pigmented bacteria (BPB) in pure cultures and in dental plaque samples obtained from human subjects with chronic periodontitis. We hypothesize that this killing effect is a result of light excitation of their endogenous porphyrins. Cultures of Prevotella intermedia and P. nigrescens were killed by 4.2 J/cm2, whereas P. melaninogenica required 21 J/cm2. Exposure to light with a fluence of 42 J/cm2 produced 99% killing of P. gingivalis. High-performance liquid chromatography demonstrated the presence of various amounts of different porphyrin molecules in BPB. The amounts of endogenous porphyrin in BPB were 267 (P. intermedia), 47 (P. nigrescens), 41 (P. melaninogenica), and 2.2 (P. gingivalis) ng/mg. Analysis of bacteria in dental plaque samples by DNA-DNA hybridization for 40 taxa before and after phototherapy showed that the growth of the four BPB was decreased by 2 and 3 times after irradiation at energy fluences of 4.2 and 21 J/cm2, respectively, whereas the growth of the remaining 36 microorganisms was decreased by 1.5 times at both energy fluences. The present study suggests that intraoral light exposure may be used to control BPB growth and possibly benefit patients with periodontal disease.