RESUMO
Historically, micro-computed tomography (µCT) has been considered unsuitable for histologic analysis of unstained formalin-fixed, paraffin-embedded soft tissue biopsy specimens because of a lack of image contrast between the tissue and the paraffin. However, we recently demonstrated that µCT can successfully resolve microstructural detail in routinely prepared tissue specimens. Herein, we illustrate how µCT imaging of standard formalin-fixed, paraffin-embedded biopsy specimens can be seamlessly integrated into conventional histology workflows, enabling nondestructive three-dimensional (3D) X-ray histology, the use and benefits of which we showcase for the exemplar of human lung biopsy specimens. This technology advancement was achieved through manufacturing a first-of-kind µCT scanner for X-ray histology and developing optimized imaging protocols, which do not require any additional sample preparation. 3D X-ray histology allows for nondestructive 3D imaging of tissue microstructure, resolving structural connectivity and heterogeneity of complex tissue networks, such as the vascular network or the respiratory tract. We also demonstrate that 3D X-ray histology can yield consistent and reproducible image quality, enabling quantitative assessment of a tissue's 3D microstructures, which is inaccessible to conventional two-dimensional histology. Being nondestructive, the technique does not interfere with histology workflows, permitting subsequent tissue characterization by means of conventional light microscopy-based histology, immunohistochemistry, and immunofluorescence. 3D X-ray histology can be readily applied to a plethora of archival materials, yielding unprecedented opportunities in diagnosis and research of disease.
Assuntos
Imageamento Tridimensional , Pneumopatias/diagnóstico por imagem , Pulmão/diagnóstico por imagem , Microtomografia por Raio-X , HumanosRESUMO
The design of nanoparticulate systems which can perform multiple synergistic functions in cells with high specificity and selectivity is of great importance in applications. Here we combine recent advances in DNA-gold nanoparticle self-assembly and sensing to develop gold nanoparticle dimers that are able to perform multiplexed synergistic functions within a cellular environment. These dimers can sense two mRNA targets and simultaneously or independently deliver one or two DNA-intercalating anticancer drugs (doxorubicin and mitoxantrone) in live cells. Our study focuses on the design of sophisticated nanoparticle assemblies with multiple and synergistic functions that have the potential to advance sensing and drug delivery in cells.
Assuntos
Antineoplásicos/farmacologia , Doxorrubicina/farmacologia , Sistemas de Liberação de Medicamentos , Ouro/química , Nanopartículas Metálicas/química , Mitoxantrona/farmacologia , RNA Mensageiro/efeitos dos fármacos , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Dimerização , Doxorrubicina/química , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Mitoxantrona/química , RNA Mensageiro/genéticaRESUMO
Lung histopathology is currently based on the analysis of 2D sections of tissue samples. The use of microfocus X-ray-computed tomography imaging of unstained soft tissue can provide high-resolution 3D image datasets in the range of 2-10 µm without affecting the current diagnostic workflow. Important details of structural features such as the tubular networks of airways and blood vessels are contained in these datasets but are difficult and time-consuming to identify by manual image segmentation. Providing 3D structures permits a better understanding of tissue functions and structural interrelationships. It also provides a more complete picture of heterogeneous samples. In addition, 3D analysis of tissue structure provides the potential for an entirely new level of quantitative measurements of this structure that have previously been based only on extrapolation from 2D sections. In this paper, a workflow for segmenting such 3D images semi-automatically has been created using and extending the ImageJ open-source software and key steps of the workflow have been integrated into a new ImageJ plug-in called LungJ. Results indicate an improved workflow with a modular organization of steps facilitating the optimization for different sample and scan properties with expert input as required. This allows for incremental and independent optimization of algorithms leading to faster segmentation. Representation of the tubular networks in samples of human lung, building on those segmentations, has been demonstrated using this approach.
Assuntos
Imageamento Tridimensional/métodos , Neoplasias Pulmonares/patologia , Pulmão/diagnóstico por imagem , Pulmão/patologia , Reconhecimento Automatizado de Padrão/métodos , Tomografia Computadorizada por Raios X/métodos , Idoso , Humanos , Pulmão/ultraestrutura , Neoplasias Pulmonares/diagnóstico por imagem , Neoplasias Pulmonares/ultraestrutura , Masculino , Fluxo de Trabalho , Raios XRESUMO
Respiratory epithelium is a highly integrated structure that efficiently protects lungs from extrinsic irritants thanks to rapid repair of the wound. The repair is a complex process that requires coordinated expression of networks of genes. Plausible regulators of this process are microRNAs. We investigated whether global miRNA silencing influences the epithelial repair, and whether changes in miRNA expression profile during repair are similar between two bronchial epithelial cell cultures: differentiated and undifferentiated cells. Two bronchial cell types were used:16HBE14o- and NHBE. Transfection was performed with siRNAs against Drosha and Dicer. For miRNA profiling, non-transfected cells were cultured until confluent and harvested for RNA isolation at baseline (cells before wounding) and at different time post-wounding (8, 16, 24, and 48 h). MicroRNA expression profiling was performed using TaqMan Array Human MicroRNA Card A. Target prediction was done in miRNA body map, and pathway analysis using DAVID. Cells with downregulated Drosha and Dicer demonstrated a significantly delayed wound repair in comparison to control in both cell lines. MiRNA expression profiling revealed that ten miRNAs exhibited significant changes over time after cell injury. These genes showed a similar expression pattern in both cell lines. The predicted targets of these miRNAs were then clustered by pathway analysis into six biological groups related to wound repair. Silencing of global miRNA expression confirmed that miRNAs are crucial for airway epithelial repair. Moreover, epithelial cells of two different origins demonstrated some similarities in miRNA expression pattern during wound repair, independent of differentiation state.
Assuntos
Diferenciação Celular , Células Epiteliais/citologia , Perfilação da Expressão Gênica , MicroRNAs/genética , Cicatrização/genética , Linhagem Celular , RNA Helicases DEAD-box/genética , Regulação para Baixo , Inativação Gênica , Humanos , Ribonuclease III/genética , TransfecçãoRESUMO
The design of nanoparticles that can selectively perform multiple roles is of utmost importance for the development of the next generation of nanoparticulate drug delivery systems. So far most research studies are focused on the customization of nanoparticulate carriers to maximize their drug loading, enhance their optical signature for tracking in cells or provide photo-responsive effects for therapeutic purposes. However, a vital requirement of the new generation of drug carriers must be the ability to deliver their payload selectively only to cells of interest rather than the majority of various cells in the vicinity. Here we show for the first time a new design of nanoparticulate drug carriers that can specifically distinguish different cell types based on their mRNA signature. These nanoparticles sense and efficiently kill model tumour cells by the delivery of an anti-cancer drug but retain their payload in cells lacking the specific mRNA target.
Assuntos
Antineoplásicos/administração & dosagem , Portadores de Fármacos , Nanopartículas , RNA Mensageiro/genética , Linhagem Celular , Humanos , Mesoderma/citologiaRESUMO
BACKGROUND: The asthma gene PCDH1 encodes Protocadherin-1, a putative adhesion molecule of unknown function expressed in the airway epithelium. Here, we characterize the localization, differential expression, homotypic adhesion specificity and function of PCDH1 in airway epithelial cells in asthma. METHODS: We performed confocal fluorescence microscopy to determine subcellular localization of PCDH1 in 16HBE cells and primary bronchial epithelial cells (PBECs) grown at air-liquid interface. Next, to compare PCDH1 expression and localization in asthma and controls we performed qRT-PCR and fluorescence microscopy in PBECs and immunohistochemistry on airway wall biopsies. We examined homotypic adhesion specificity of HEK293T clones overexpressing fluorescently tagged-PCDH1 isoforms. Finally, to evaluate the role for PCDH1 in epithelial barrier formation and repair, we performed siRNA knockdown-studies and measured epithelial resistance. RESULTS: PCDH1 localized to the cell membrane at cell-cell contact sites, baso-lateral to adherens junctions, with increasing expression during epithelial differentiation. No differences in gene expression or localization of PCDH1 isoforms expressing the extracellular domain were observed in either PBECs or airway wall biopsies between asthma patients and controls. Overexpression of PCDH1 mediated homotypic interaction, whereas downregulation of PCDH1 reduced epithelial barrier formation, and impaired repair after wounding. CONCLUSIONS: In conclusion, PCDH1 is localized to the cell membrane of bronchial epithelial cells baso-lateral to the adherens junction. Expression of PCDH1 is not reduced nor delocalized in asthma even though PCDH1 contributes to homotypic adhesion, epithelial barrier formation and repair.
Assuntos
Asma/metabolismo , Brônquios/citologia , Caderinas/genética , Caderinas/metabolismo , Membrana Celular/metabolismo , Células Epiteliais/metabolismo , Junções Aderentes/metabolismo , Idoso , Asma/genética , Brônquios/metabolismo , Adesão Celular , Células Epiteliais/citologia , Feminino , Regulação da Expressão Gênica , Células HEK293 , Humanos , Masculino , Pessoa de Meia-Idade , Protocaderinas , Adulto JovemRESUMO
Nasal drug administration is a promising alternative to oral and parenteral administration for both local and systemic delivery of drugs. The benefits include its noninvasive nature, rapid absorption, and circumvention of first pass metabolism. Hence, the use of an in vitro model using human primary nasal epithelial cells could be key to understanding important functions and parameters of the respiratory epithelium. This model will enable investigators to address important and original research questions using a biologically relevant in vitro platform that mimics the in vivo nasal epithelial physiology. The purpose of this study was to establish, systematically characterize, and validate the use of a primary human nasal epithelium model cultured at the air-liquid interface for the study of inflammatory responses and drug transport and to simultaneously quantify drug effects on ciliary activity.
Assuntos
Células Epiteliais/fisiologia , Mucosa Nasal/fisiologia , Preparações Farmacêuticas/administração & dosagem , Mucosa Respiratória/fisiologia , Administração Intranasal/métodos , Adulto , Técnicas de Cultura de Células/métodos , Células Cultivadas , Sistemas de Liberação de Medicamentos/métodos , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
BACKGROUND: Airway NO synthase (NOS) isoenzymes are responsible for rapid and localised nitric oxide (NO) production and are expressed in airway epithelium. We sought to determine the localisation of neuronal NOS (nNOS) in airway epithelium due to the paucity of evidence. METHODS AND RESULTS: Sections of healthy human bronchial tissue in glycol methacrylate resin and human nasal polyps in paraffin wax were immunohistochemically labelled and reproducibly demonstrated nNOS immunoreactivity, particularly at the proximal portion of cilia; this immunoreactivity was blocked by a specific nNOS peptide fragment. Healthy human epithelial cells differentiated at an air-liquid interface (ALI) confirmed the presence of all three NOS isoenzymes by immunofluorescence labelling. Only nNOS immunoreactivity was specific to the ciliary axonemeand co-localised with the cilia marker ß-tubulin in the proximal part of the ciliary axoneme. CONCLUSIONS: We report a novel localisation of nNOS at the proximal portion of cilia in airway epithelium and conclude that its independent and local regulation of NO levels is crucial for normal cilia function.
Assuntos
Cílios/enzimologia , Óxido Nítrico Sintase Tipo I/metabolismo , Mucosa Respiratória/enzimologia , Brônquios/química , Brônquios/enzimologia , Células Cultivadas , Cílios/química , Cílios/metabolismo , Humanos , Imuno-Histoquímica , Pólipos Nasais/química , Pólipos Nasais/enzimologia , Óxido Nítrico Sintase Tipo I/química , Mucosa Respiratória/química , Mucosa Respiratória/citologia , Mucosa Respiratória/metabolismoRESUMO
BACKGROUND: The diagnosis of primary ciliary dyskinesia (PCD) requires the analysis of ciliary function and ultrastructure. Diagnosis can be complicated by secondary effects on cilia such as damage during sampling, local inflammation or recent infection. To differentiate primary from secondary abnormalities, re-analysis of cilia following culture and re-differentiation of epithelial cells at an air-liquid interface (ALI) aids the diagnosis of PCD. However changes in ciliary beat pattern of cilia following epithelial cell culture has previously been described, which has brought the robustness of this method into question. This is the first systematic study to evaluate ALI culture as an aid to diagnosis of PCD in the light of these concerns. METHODS: We retrospectively studied changes associated with ALI-culture in 158 subjects referred for diagnostic testing at two PCD centres. Ciliated nasal epithelium (PCD n = 54; non-PCD nâ 111) was analysed by high-speed digital video microscopy and transmission electron microscopy before and after culture. RESULTS: Ciliary function was abnormal before and after culture in all subjects with PCD; 21 PCD subjects had a combination of static and uncoordinated twitching cilia, which became completely static following culture, a further 9 demonstrated a decreased ciliary beat frequency after culture. In subjects without PCD, secondary ciliary dyskinesia was reduced. CONCLUSIONS: The change to ciliary phenotype in PCD samples following cell culture does not affect the diagnosis, and in certain cases can assist the ability to identify PCD cilia.
Assuntos
Cílios/genética , Transtornos da Motilidade Ciliar/genética , Síndrome de Kartagener/genética , Ar , Técnicas de Cultura de Células , Células Cultivadas , Cílios/fisiologia , Transtornos da Motilidade Ciliar/diagnóstico , Células Epiteliais/citologia , Humanos , Síndrome de Kartagener/diagnóstico , Microscopia Eletrônica de Transmissão , Microscopia de Vídeo , Mucosa Nasal , Fenótipo , Estudos RetrospectivosRESUMO
Asthma exacerbations are among the most frequent causes of hospitalization during childhood, but the underlying mechanisms are poorly understood. We performed a genome-wide association study of a specific asthma phenotype characterized by recurrent, severe exacerbations occurring between 2 and 6 years of age in a total of 1,173 cases and 2,522 controls. Cases were identified from national health registries of hospitalization, and DNA was obtained from the Danish Neonatal Screening Biobank. We identified five loci with genome-wide significant association. Four of these, GSDMB, IL33, RAD50 and IL1RL1, were previously reported as asthma susceptibility loci, but the effect sizes for these loci in our cohort were considerably larger than in the previous genome-wide association studies of asthma. We also obtained strong evidence for a new susceptibility gene, CDHR3 (encoding cadherin-related family member 3), which is highly expressed in airway epithelium. These results demonstrate the strength of applying specific phenotyping in the search for asthma susceptibility genes.
Assuntos
Asma/genética , Caderinas/genética , Predisposição Genética para Doença , Proteínas de Membrana/genética , Hidrolases Anidrido Ácido , Asma/etiologia , Proteínas Relacionadas a Caderinas , Caderinas/química , Caderinas/metabolismo , Estudos de Casos e Controles , Criança , Pré-Escolar , Cromossomos Humanos Par 17 , Enzimas Reparadoras do DNA/genética , Proteínas de Ligação a DNA/genética , Dinamarca , Feminino , Estudo de Associação Genômica Ampla , Humanos , Proteína 1 Semelhante a Receptor de Interleucina-1 , Interleucina-33 , Interleucinas/genética , Masculino , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Modelos Moleculares , Proteínas de Neoplasias/genética , Polimorfismo de Nucleotídeo Único , Conformação Proteica , Receptores de Superfície Celular/genéticaRESUMO
BACKGROUND: Airway epithelial cells provide a protective barrier against environmental particles including potential pathogens. Epithelial repair in response to tissue damage is abnormal in asthmatic airway epithelium in comparison to the repair of normal epithelium after damage. The complex mechanisms coordinating the regulation of the processes involved in wound repair requires the phased expression of networks of genes. Small non-coding RNA molecules termed microRNAs (miRNAs) play a critical role in such coordinated regulation of gene expression. We aimed to establish if the phased expression of specific miRNAs is correlated with the repair of mechanically induced damage to the epithelium. METHODS: To investigate the possible involvement of miRNA in epithelial repair, we analyzed miRNA expression profiles during epithelial repair in a cell culture model using TaqMan-based quantitative real-time PCR in a TaqMan Low Density Array format. The expression of 754 miRNA genes at seven time points in a 48-hour period during the wound repair process was profiled using the bronchial epithelial cell line 16HBE14o- growing in monolayer. RESULTS: The expression levels of numerous miRNAs were found to be altered during the wound repair process. These miRNA genes were clustered into 3 different patterns of expression that correlate with the further regulation of several biological pathways involved in wound repair. Moreover, it was observed that expression of some miRNA genes were significantly altered only at one time point, indicating their involvement in a specific stage of the epithelial wound repair. CONCLUSIONS: In summary, miRNA expression is modulated during the normal repair processes in airway epithelium in vitro suggesting a potential role in regulation of wound repair.
Assuntos
Brônquios/citologia , Células Epiteliais/citologia , Perfilação da Expressão Gênica , MicroRNAs/genética , Cicatrização/genética , Células Cultivadas , Regulação para Baixo , Regulação da Expressão Gênica , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Transdução de Sinais/genética , Fatores de Tempo , Regulação para CimaRESUMO
Environmental particulates impact first on airway epithelium, whereas circulating infiltrating cells are recruited through the underlying endothelium. An effective cellular immune response requires coordination between endothelium and epithelium. The authors have developed a bilayer culture model consisting of human bronchial epithelial derived cells (16HBE 14o-) and human umbilical vein endothelial cells (HUVECs) cultured as confluent layers on either side of a porous membrane. Confocal microscopy with epithelial and endothelial-specific antibodies showed segregated cell layers. By scanning and transmission electron microscopy, both cell types are polarized and tight junctions formed at the apical interface between cells. Epithelial cells grown in a bilayer showed significantly increased transepithelial resistance (TER) of 2260 +/- 64 Omega.cm(2) compared to epithelial or endothelial monolayers alone (1400 +/- 70 or 80 +/- 12 Omega.cm(2), respectively). This reflected decreased permeability and was unrelated to cell density or height. Increased TER coincided with increased occludin mRNA and protein in the epithelial cell layer as determined by polymerase chain reaction (PCR) and immunoblotting. Conditioned medium showed that decreased permeability was mediated by soluble endothelial-derived factor(s). This model reflects the in vivo relationship of human airway endothelial cells and epithelial cells. Altered tight junction permeability in cocultures indicates that these cells can work together as an active part of the mucosal barrier.
Assuntos
Comunicação Celular/fisiologia , Células Endoteliais/metabolismo , Células Epiteliais/metabolismo , Mucosa Respiratória/citologia , Junções Íntimas/metabolismo , Brônquios/citologia , Polaridade Celular , Técnicas de Cocultura , Endotélio Vascular/citologia , Células Epiteliais/química , Humanos , Proteínas de Membrana/análise , Proteínas de Membrana/genética , Microscopia Eletrônica , Ocludina , Permeabilidade , RNA Mensageiro/análiseRESUMO
The effective repair of damage to the airway epithelium is essential to maintain the ability to exclude airborne particulates and protect against potential pathogens. Carbohydrates on the cell surface have an important role in cell-cell and cell substrate interactions. Using a model of repair with airway epithelial-derived cells of the 16HBE 14o(-) cell line, we have examined the effect of the Aleuria aurantia lectin (AAL), which binds very selectively to alpha(1,6)-linked fucose residues. Addition of unconjugated or FITC-labeled AAL reduced the rate of epithelial repair to approximately one-third of control values as measured by image analysis while cell viability was maintained. Pulse labeling with AAL-FITC for 30 min followed by incubation in AAL-free medium caused similar inhibition of repair but could be reversed by addition of fucose up to 7 h after AAL removal. By confocal microscopy, AAL binding was found to be on the apical, but not basolateral, surfaces of cells, and internalization of the labeled lectin was seen. Preincubation of the lectin with fucose prevented this effect. Ulex europeaus I lectin, which is also fucose specific, resulted in similar binding to the cells and internalization, but it did not affect the speed of the repair process. We conclude that alpha(1,6)-fucose binding sites play an important role in epithelial repair. Better understanding of this process will provide a deeper insight into the crucial mechanisms of epithelial repair.
Assuntos
Epitélio/efeitos dos fármacos , Epitélio/fisiologia , Lectinas/farmacologia , Cicatrização/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Humanos , Ligação Proteica/efeitos dos fármacos , Fatores de TempoRESUMO
In airways, the cell surface molecule CD44 is upregulated on bronchial epithelial cells in areas of damage. We have shown that a blocking standard CD44 (CD44s) antibody caused a 77% (+/- 19%) inhibition of cell migration at 3 h after mechanical damage and decreased epithelial cell repair of cells grown on cell culture filter inserts. With the use of primary human bronchial epithelial cells and the bronchial epithelial cell line 16HBE 14o-, a CD44s antibody inhibited >95% (P < 0.01) of cell binding to hyaluronic acid (HA). The cytokines TNF-alpha, IFN-gamma, IL-1 beta, and IL-4 stimulated a 2- to 3.5-fold increase in CD44-dependent cell binding to HA. IFN-gamma treatment did not increase CD44 expression as assessed by flow cytometry, although phorbol myristate acetate treatment did. This indicates that IFN-gamma-induced cell binding to HA did not require increased CD44 expression. These data indicate that CD44 is important for bronchial epithelial cell binding to HA and that cytokines known to be expressed in inflammation can increase HA binding independently of the level of CD44 expression.
Assuntos
Antineoplásicos/farmacologia , Células Epiteliais/metabolismo , Receptores de Hialuronatos/metabolismo , Interferon gama/farmacologia , Mucosa Respiratória/citologia , Adjuvantes Imunológicos/metabolismo , Adjuvantes Imunológicos/farmacologia , Anticorpos/farmacologia , Adesão Celular/fisiologia , Linhagem Celular Transformada , Movimento Celular/fisiologia , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Humanos , Ácido Hialurônico/metabolismo , Ácido Hialurônico/farmacologia , Regulação para Cima/imunologia , Cicatrização/imunologiaRESUMO
Restitution of an epithelial layer after environmental or biological damage is important to maintain the normal function of the respiratory tract. We have investigated the role of transforming growth factor (TGF)-beta isoforms in the repair of layers of 16HBE 14o(-) bronchial epithelial-derived cells after damage by multiple scoring. ELISA showed that both latent TGF-beta1 and TGF-beta2 were converted to their active forms 2 h after wounding. Time-lapse microscopy showed that the addition of TGF-beta1, but not TGF-beta2, progressively increased the rate of migration of damaged monolayers at concentrations down to 250 pg/ml. This increase was blocked by addition of a neutralizing TGF-beta1 antibody. Phase-contrast microscopy and inhibition of proliferation with mitomycin C showed that proliferation was not required for migration. These results demonstrate that conversion of latent to active TGF-beta1 and TGF-beta2 during in vitro epithelial wound repair occurs quickly and that TGF-beta1 speeds epithelial repair. A faster repair may be advantageous in preventing access of environmental agents to the internal milieu of the lung although the production of active TGF-beta molecules may augment subepithelial fibrosis.