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Background: The University of Southampton, in collaboration with the University Hospital Southampton (UHS) NHS Foundation Trust and industrial partners, has been at the forefront of developing three-dimensional (3D) imaging workflows using X-ray microfocus computed tomography (µCT) -based technology. This article presents the outcomes of these endeavours and highlights the distinctive characteristics of a µCT facility tailored explicitly for 3D X-ray Histology, with a primary focus on applications in biomedical research and preclinical and clinical studies. Methods: The UHS houses a unique 3D X-ray Histology (XRH) facility, offering a range of services to national and international clients. The facility employs specialised µCT equipment explicitly designed for histology applications, allowing whole-block XRH imaging of formalin-fixed and paraffin-embedded tissue specimens. It also enables correlative imaging by combining µCT imaging with other microscopy techniques, such as immunohistochemistry (IHC) and serial block-face scanning electron microscopy, as well as data visualisation, image quantification, and bespoke analysis. Results: Over the past seven years, the XRH facility has successfully completed over 120 projects in collaboration with researchers from 60 affiliations, resulting in numerous published manuscripts and conference proceedings. The facility has streamlined the µCT imaging process, improving productivity and enabling efficient acquisition of 3D datasets. Discussion & Conclusions: The 3D X-ray Histology (XRH) facility at UHS is a pioneering platform in the field of histology and biomedical imaging. To the best of our knowledge, it stands out as the world's first dedicated XRH facility, encompassing every aspect of the imaging process, from user support to data generation, analysis, training, archiving, and metadata generation. This article serves as a comprehensive guide for establishing similar XRH facilities, covering key aspects of facility setup and operation. Researchers and institutions interested in developing state-of-the-art histology and imaging facilities can utilise this resource to explore new frontiers in their research and discoveries.
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Air-liquid interface (ALI) culture of nasal epithelial cells is a valuable tool in the diagnosis and research of primary ciliary dyskinesia (PCD). Ex vivo samples often display secondary dyskinesia from cell damage during sampling, infection or inflammation confounding PCD diagnostic results. ALI culture enables regeneration of healthy cilia facilitating differentiation of primary from secondary ciliary dyskinesia. We describe a revised ALI culture method adopted from April 2018 across three collaborating PCD diagnostic sites, including current University Hospital Southampton COVID-19 risk mitigation measures, and present results. Two hundred and forty nasal epithelial cell samples were seeded for ALI culture and 199 (82.9%) were ciliated. Fifty-four of 83 (63.9%) ex vivo samples which were originally equivocal or insufficient provided diagnostic information following in vitro culture. Surplus basal epithelial cells from 181 nasal brushing samples were frozen in liquid nitrogen; 39 samples were ALI-cultured after cryostorage and all ciliated. The ciliary beat patterns of ex vivo samples (by high-speed video microscopy) were recapitulated, scanning electron microscopy demonstrated excellent ciliation, and cilia could be immuno-fluorescently labelled (anti-alpha-tubulin and anti-RSPH4a) in representative cases that were ALI-cultured after cryostorage. In summary, our ALI culture protocol provides high ciliation rates across three centres, minimising patient recall for repeat brushing biopsies and improving diagnostic certainty. Cryostorage of surplus diagnostic samples was successful, facilitating PCD research.
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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.
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Imageamento Tridimensional , Pneumopatias/diagnóstico por imagem , Pulmão/diagnóstico por imagem , Microtomografia por Raio-X , HumanosRESUMO
Lung lymphatics maintain fluid homoeostasis by providing a drainage system that returns fluid, cells and metabolites to the circulatory system. The 3D structure of the human pulmonary lymphatic network is essential to lung function, but it is poorly characterised. Image-based 3D mathematical modelling of pulmonary lymphatic microfluidics has been limited by the lack of accurate and representative image geometries. This is due to the microstructural similarity of the lymphatics to the blood vessel network, the lack of lymphatic-specific biomarkers, the technical limitations associated with image resolution in 3D, and sectioning artefacts present in 2D techniques. We present a method that combines lymphatic specific (D240 antibody) immunohistochemistry (IHC), optimised high-resolution X-ray microfocus computed tomography (µCT) and finite-element mathematical modelling to assess the function of human peripheral lung tissue. The initial results identify lymphatic heterogeneity within and between lung tissue. Lymphatic vessel volume fraction and fractal dimension significantly decreases away from the lung pleural surface (p < 0.001, n = 25 and p < 0.01, n = 20, respectively). Microfluidic modelling successfully shows that in lung tissue the fluid derived from the blood vessels drains through the interstitium into the lymphatic vessel network and this drainage is different in the subpleural space compared to the intralobular space. When comparing lung tissue from health and disease, human pulmonary lymphatics were significantly different across five morphometric measures used in this study (p ≤ 0.0001). This proof of principle study establishes a new engineering technology and workflow for further studies of pulmonary lymphatics and demonstrates for the first time the combination of correlative µCT and IHC to enable 3D mathematical modelling of human lung microfluidics at micrometre resolution.
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Imageamento Tridimensional/métodos , Pulmão/ultraestrutura , Vasos Linfáticos/ultraestrutura , Microfluídica/métodos , Humanos , Imageamento Tridimensional/instrumentação , Imuno-Histoquímica , Pulmão/metabolismo , Vasos Linfáticos/metabolismo , Microtomia , Modelos Biológicos , Microtomografia por Raio-XRESUMO
PURPOSE: Airway epithelium acts as a protective barrier against the particles from the inhaled air. Damage to the epithelium may result in loss of the barrier function. Epithelial repair in response to injury requires complex mechanisms, such as microRNA, small noncoding molecules, to regulate the processes involved in wound repair. We aimed to establish if the microRNA gene expression profile is altered during the airway epithelial repair in differentiated cells. METHODS: miRNA gene expression profile during the wound closure of differentiated normal human bronchial epithelium (NHBE) from one donor was analysed using quantitative real-time PCR. We have analysed the expression of 754 genes at five time points during a 48-hour period of epithelium repair using TaqMan Low Density Array. RESULTS: We found out that 233 miRNA genes were expressed in normal human bronchial epithelium. Twenty miRNAs were differentially expressed during the wound repair process, but only one (miR-455-3p) showed significance after FDR adjustment (p = 0.02). Using STEM, we have identified two clusters of several miRNA genes with similar expression profile. Pathway enrichment analysis showed several significant signaling pathways altered during repair, mainly involved in cell cycle regulation, proliferation, migration, adhesion, and transcription regulation. CONCLUSIONS: miRNA expression profile is altered during airway epithelial repair of differentiated cells from one donor in response to mechanical injury in vitro, suggesting their potential role in wound repair.
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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.
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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.
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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.
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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.
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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.
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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
In idiopathic pulmonary fibrosis (IPF), the fibroblast focus is a key histological feature representing active fibroproliferation. On standard 2D pathologic examination, fibroblast foci are considered small, distinct lesions, although they have been proposed to form a highly interconnected reticulum as the leading edge of a "wave" of fibrosis. Here, we characterized fibroblast focus morphology and interrelationships in 3D using an integrated micro-CT and histological methodology. In 3D, fibroblast foci were morphologically complex structures, with large variations in shape and volume (range, 1.3 × 104 to 9.9 × 107 µm3). Within each tissue sample numerous multiform foci were present, ranging from a minimum of 0.9 per mm3 of lung tissue to a maximum of 11.1 per mm3 of lung tissue. Each focus was an independent structure, and no interconnections were observed. Together, our data indicate that in 3D fibroblast foci form a constellation of heterogeneous structures with large variations in shape and volume, suggesting previously unrecognized plasticity. No evidence of interconnectivity was identified, consistent with the concept that foci represent discrete sites of lung injury and repair.
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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.
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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: Understanding the three-dimensional (3-D) micro-architecture of lung tissue can provide insights into the pathology of lung disease. Micro computed tomography (µCT) has previously been used to elucidate lung 3D histology and morphometry in fixed samples that have been stained with contrast agents or air inflated and dried. However, non-destructive microstructural 3D imaging of formalin-fixed paraffin embedded (FFPE) tissues would facilitate retrospective analysis of extensive tissue archives of lung FFPE lung samples with linked clinical data. METHODS: FFPE human lung tissue samples (n = 4) were scanned using a Nikon metrology µCT scanner. Semi-automatic techniques were used to segment the 3D structure of airways and blood vessels. Airspace size (mean linear intercept, Lm) was measured on µCT images and on matched histological sections from the same FFPE samples imaged by light microscopy to validate µCT imaging. RESULTS: The µCT imaging protocol provided contrast between tissue and paraffin in FFPE samples (15 mm x 7 mm). Resolution (voxel size 6.7 µm) in the reconstructed images was sufficient for semi-automatic image segmentation of airways and blood vessels as well as quantitative airspace analysis. The scans were also used to scout for regions of interest, enabling time-efficient preparation of conventional histological sections. The Lm measurements from µCT images were not significantly different to those from matched histological sections. CONCLUSION: We demonstrated how non-destructive imaging of routinely prepared FFPE samples by laboratory µCT can be used to visualize and assess the 3D morphology of the lung including by morphometric analysis.
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Imageamento Tridimensional/métodos , Pulmão/diagnóstico por imagem , Microtomografia por Raio-X/métodos , Formaldeído , Humanos , Pulmão/irrigação sanguínea , Inclusão em Parafina , Fixação de TecidosRESUMO
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.
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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.
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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.
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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.
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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
Nitric oxide is continually synthesised in the respiratory epithelium and is upregulated in response to infection or inflammation. Primary ciliary dyskinesia (PCD) is characterised by recurrent sinopulmonary infections due to impaired mucociliary clearance. Despite chronic infections, nasal nitric oxide in such patients is markedly reduced and is used as a screening test for this condition. These low levels were first described >15 yrs ago but the underlying mechanisms have yet to be fully elucidated. We review epithelial nitric oxide synthesis, release and measurement in the upper airways with particular reference to PCD. The key hypotheses that have been proposed to explain the low nitric oxide levels in this condition are explored and the potential benefits of augmenting airway nitric oxide levels are considered. Further work in these patients clarifying both whether the respiratory epithelium is able to biosynthesise normal levels of nitric oxide and the role played by abnormalities in the anatomy of the paranasal sinuses is essential. While nitric oxide augmentation is unlikely to be beneficial in common PCD phenotypes, it has potential in the treatment of secondary dyskinesias and may also improve treatment of bacterial infections, particularly where biofilms are implicated.
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Síndrome de Kartagener/metabolismo , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico/metabolismo , Mucosa Respiratória/metabolismo , Testes Respiratórios , Humanos , Óxido Nítrico Sintase/genéticaRESUMO
Primary ciliary dyskinesia (PCD) is an inherited disorder causing significant upper and lower respiratory tract morbidity and impaired fertility. Half of PCD patients show abnormal situs. Human disease loci have been identified but a mouse model without additional deleterious defects is elusive. The inversus viscerum mouse, mutated at the outer arm dynein heavy chain 11 locus (Dnahc11) is a known model of heterotaxy. We demonstrated immotile tracheal cilia with normal ultrastructure and reduced sperm motility in the Dnahc11(iv) mouse. This is accompanied by gross rhinitis, sinusitis, and otitis media, all indicators of human PCD. Strikingly, age-related progression of the disease is evident. The Dnahc11(iv) mouse is robust, lacks secondary defects, and requires no intervention to precipitate the phenotype. Together these findings show the Dnahc11(iv) mouse to be an excellent model of many aspects of human PCD. Mutation of the homologous human locus has previously been associated with hyperkinetic tracheal cilia in PCD. Two PCD patients with normal ciliary ultrastructure, one with immotile and one with hyperkinetic cilia were found to carry DNAH11 mutations. Three novel DNAH11 mutations were detected indicating that this gene should be investigated in patients with normal ciliary ultrastructure and static, as well as hyperkinetic cilia.