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Comparison of FcRn- and pIgR-mediated transport in MDCK cells by fluorescence confocal microscopy.

ABSTRACT: Protein delivery across polarized epithelia is controlled by receptor-mediated transcytosis. Many studies have examined basolateral-to-apical trafficking of polymeric IgA (pIgA) by the polymeric immunoglobulin receptor (pIgR). Less is known about apical-to-basolateral transcytosis, the direction the neonatal Fc receptor (FcRn) transports maternal IgGs across intestinal epithelia. To compare apical-to-basolateral and basolateral-to-apical transcytosis, we co-expressed FcRn and pIgR in Madin-Darby canine kidney (MDCK) cells and used pulse-chase experiments with confocal microscopy to examine transport of apically applied IgG Fcgamma and basolaterally applied pIgA. Fcgamma and pIgA trafficking routes were initially separate but intermixed at later chase times. Fcgamma was first localized near the apical surface, but became more equally distributed across the cell, consistent with concomitant transcytosis and recycling. By contrast, pIgA transport was strongly unidirectional: pIgA shifted from near the basolateral surface to an apical location with increasing time. Some Fcgamma and pIgA fluorescence colocalized in early (EEA1-positive), recycling (Rab11a-positive), and transferrin (Tf)-positive common/basolateral recycling endosomes. Fcgamma became more enriched in Tf-positive endosomes with time, whereas pIgA was sorted from these compartments. Live-cell imaging revealed that vesicles containing Fcgamma or pIgA shared similar mobility characteristics and were equivalently affected by depolymerizing microtubules, indicating that both trafficking routes depended to roughly the same extent on intact microtubules.

SUBMITTER: Jerdeva GV

PROVIDER: S-EPMC2975666 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Importance:Strategies to procure high-quality core-needle biopsy (CNB) specimens are critical for making basic tissue diagnoses and for ancillary testing. Objectives:To investigate acquisition of fluorescence confocal microscopy (FCM) images of interventional radiology (IR)-guided CNB in real time in the radiology suite and to compare the accuracy of FCM diagnoses with those of hematoxylin-eosin (H&E)-stained CNB sections. Design, Setting, and Participants:In this diagnostic study, FCM imaging of IR-guided CNBs was performed in the radiology suite at a major cancer center for patients with an imaging abnormality from August 1, 2016, to April 30, 2019. The time taken to acquire FCM images and the quality of FCM images based on percentage of interpretable tissue with optimal resolution was recorded. The FCM images were read by 2 pathologists and categorized as nondiagnostic, benign/atypical, or suspicious/malignant; these diagnoses were compared with those made using H&E-stained tissue sections. Cases with discrepant diagnosis were reassessed by the pathologists together for a consensus diagnosis. Data were analyzed from June 3 to July 19, 2019. Interventions:Each IR-guided CNB was stained with 0.6mM acridine orange, subjected to FCM imaging, and then processed to generate H&E-stained sections. Main Outcomes and Measures:Mean time taken for acquisition of FCM images, quality of FCM images based on interpretable percentage of the image, and accuracy of diagnostic categorization based on FCM images compared with H&E-stained sections. Results:A total of 105 patients (57 male [54.3%]; mean [SD] age, 63 [13] years) underwent IR-guided CNBs in a mean (SD) of 7 (2) minutes each. The FCM images showed at least 20% of the tissue with optimal quality in 101 CNB specimens (96.2%). The FCM images were accurately interpreted by the 2 pathologists in 100 of 105 cases (95.2%) (2 false-positive and 3 false-negative) and 90 of 105 cases (85.7%) (6 false-positive and 9 false-negative). A reassessment of 14 discordant diagnoses resulted in consensus diagnoses that were accurate in 101 of 105 cases (96.2%) (1 false-positive and 3 false-negative). Conclusions and Relevance:The ease of acquisition of FCM images of acceptable quality and the high accuracy of the diagnoses suggest that FCM may be useful for rapid evaluation of IR-guided CNBs. This approach warrants further investigation.

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Comparison of FcRn- and pIgR-mediated transport in MDCK cells by fluorescence confocal microscopy.

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