RESUMO
BACKGROUND AND AIMS: Endoscopic surveillance of Barrett's esophagus (BE) by white light examination is insufficient to diagnose dysplastic change. In this work, we describe an optical imaging method to obtain high-resolution cross-sectional imaging using a paddle-shaped probe affixed to the endoscope tip. METHODS: We integrated Optical Coherence Tomography (OCT), an optical imaging method that produces cross-sectional images, into a paddle probe attached to video endoscope. We acquired images of esophageal epithelium from patients undergoing routine upper GI endoscopy. Images were classified by a reviewer blinded to patient identity and condition, and these results were compared with clinical diagnosis. RESULTS: We successfully captured epithelial OCT images from 30 patients and identified features consistent with both squamous epithelium and Barrett's esophagus. Our blinded image reviewer classified BE versus non-BE with 91.5% accuracy (65/71 image regions), including sensitivity of 84.6% for BE (11/13) and a specificity of 93.1% (54/58). However, in 16 patients, intubation of the probe into the esophagus could not be achieved. CONCLUSIONS: A paddle probe is a feasible imaging format for acquiring cross-sectional OCT images from the esophagus and can provide a structural assessment of BE and non-BE tissue. Probe form factor is the current limiting obstacle, but could be addressed by further miniaturization.
Assuntos
Esôfago de Barrett , Neoplasias Esofágicas , Esôfago de Barrett/diagnóstico por imagem , Endoscópios , Endoscopia do Sistema Digestório , Esofagoscopia/métodos , Humanos , Tomografia de Coerência Óptica/métodosRESUMO
Optical coherence tomography (OCT) is used for diagnosis of esophageal diseases such as Barrett's esophagus. Given the large volume of OCT data acquired, automated analysis is needed. Here we propose a bilateral connectivity-based neural network for in vivo human esophageal OCT layer segmentation. Our method, connectivity-based CE-Net (Bicon-CE), defines layer segmentation as a combination of pixel connectivity modeling and pixel-wise tissue classification. Bicon-CE outperformed other widely used neural networks and reduced common topological prediction issues in tissues from healthy patients and from patients with Barrett's esophagus. This is the first end-to-end learning method developed for automatic segmentation of the epithelium in in vivo human esophageal OCT images.
RESUMO
Nucleotides within the airway surface liquid promote fluid secretion via activation of airway epithelial purinergic receptors. ATP is stored within and released from mucin granules as co-cargo with mucins, but the mechanism by which ATP, and potentially other nucleotides, enter the lumen of mucin granules is not known. We assessed the contribution of the recently identified SLC17A9 vesicle nucleotide transporter (VNUT) to the nucleotide availability within isolated mucin granules and further examined the involvement of VNUT in mucin granule secretion-associated nucleotide release. RT-PCR and Western blot analyses indicated that VNUT is abundantly expressed in airway epithelial goblet-like Calu-3 cells, migrating as a duplex with apparent mobility of 55 and 60 kDa. Subcellular fractionation studies indicated that VNUT55 was associated with high-density mucin granules, whereas VNUT60 was associated with low-density organelles. Immunofluorescence studies showed that recombinant VNUT localized to mucin granules and other organelles. Mucin granules isolated from VNUT short hairpin RNA-expressing cells exhibited a marked reduction of ATP, ADP, AMP, and UTP levels within granules. Ca(2+)-regulated vesicular ATP release was markedly reduced in these cells, but mucin secretion was not affected. These results suggest that VNUT is the relevant nucleotide transporter responsible for the uptake of cytosolic nucleotides into mucin granules. By controlling the entry of nucleotides into mucin granules, VNUT contributes to the release of purinergic signaling molecules necessary for the proper hydration of co-released mucins.
Assuntos
Células Caliciformes/metabolismo , Proteínas de Transporte de Nucleotídeos/metabolismo , Nucleotídeos/metabolismo , Sistema Respiratório/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Difosfato de Adenosina/biossíntese , Monofosfato de Adenosina/biossíntese , Trifosfato de Adenosina/biossíntese , Trifosfato de Adenosina/metabolismo , Transporte Biológico , Linhagem Celular , Grânulos Citoplasmáticos/metabolismo , Humanos , Mucinas/genética , Proteínas de Transporte de Nucleotídeos/biossíntese , RNA Interferente Pequeno , Vesículas Secretórias/metabolismo , Uridina Trifosfato/biossínteseRESUMO
Mucin secretion is an innate defence mechanism, which is noxiously upregulated in obstructive lung diseases (e.g. chronic obstructive pulmonary disease (COPD), cystic fibrosis and asthma). Mucin granule exocytosis is regulated by specific protein complexes, but the SNARE exocytotic core has not been defined in airway goblet cells. In this study, we identify VAMP8 as one of the SNAREs regulating mucin granule exocytosis. VAMP8 mRNA was present in human airway and lung epithelial cells, and deep-sequencing and expression analyses of airway epithelial cells revealed that VAMP8 transcripts were expressed at 10 times higher levels than other VAMP mRNAs. In human airway epithelial cell cultures and freshly excised tissues, VAMP8 immunolocalised mainly to goblet cell mucin granules. The function of VAMP8 in airway mucin secretion was tested by RNA interference techniques. Both VAMP8 short interfering RNAs (siRNAs) and short hairpin RNAs (shRNAs) reduced mucin secretion induced by PAR agonists, neutrophil elastase and ATP in two airway epithelial cell culture models. Notably, basal (non-agonist elicited) mucin secretion was also reduced in these experiments. VAMP8 knockdown was also effective in decreasing mucin secretion in airway epithelial cell cultures with induced mucous metaplasia/mucin hypersecretion. Unlike VAMP8 silencing, knockdown of VAMP2 or VAMP3 did not affect mucin secretion. Importantly, in VAMP8 knock-out (KO) mice with IL-13-induced mucous metaplasia, mucin content in the bronchoalveolar lavage (BAL) and ATP-stimulated mucin secretion in the trachea were reduced compared to WT-matched littermates. Our data indicate that VAMP8 is an essential SNARE in airway mucin granule exocytosis. Reduction of VAMP8 activity/expression may provide a novel therapeutic target to ameliorate airway mucus obstruction in lung diseases.