Imaging intact human organs from the organ to the cellular scale in three dimensions is a goal of biomedical imaging. To meet this challenge, we developed hierarchical phase-contrast tomography ...(HiP-CT), an X-ray phase propagation technique using the European Synchrotron Radiation Facility (ESRF)'s Extremely Brilliant Source (EBS). The spatial coherence of the ESRF-EBS combined with our beamline equipment, sample preparation and scanning developments enabled us to perform non-destructive, three-dimensional (3D) scans with hierarchically increasing resolution at any location in whole human organs. We applied HiP-CT to image five intact human organ types: brain, lung, heart, kidney and spleen. HiP-CT provided a structural overview of each whole organ followed by multiple higher-resolution volumes of interest, capturing organotypic functional units and certain individual specialized cells within intact human organs. We demonstrate the potential applications of HiP-CT through quantification and morphometry of glomeruli in an intact human kidney and identification of regional changes in the tissue architecture in a lung from a deceased donor with coronavirus disease 2019 (COVID-19).
Imaging across different scales is essential for understanding healthy organ morphology and pathophysiological changes. The macro- and microscale three-dimensional morphology of large samples, ...including intact human organs, is possible with X-ray microtomography (using laboratory or synchrotron sources). Preparation of large samples for high-resolution imaging, however, is challenging due to limitations such as sample shrinkage, insufficient contrast, movement of the sample and bubble formation during mounting or scanning. Here, we describe the preparation, stabilization, dehydration and mounting of large soft-tissue samples for X-ray microtomography. We detail the protocol applied to whole human organs and hierarchical phase-contrast tomography at the European Synchrotron Radiation Facility, yet it is applicable to a range of biological samples, including complete organisms. The protocol enhances the contrast when using X-ray imaging, while preventing sample motion during the scan, even with different sample orientations. Bubbles trapped during mounting and those formed during scanning (in the case of synchrotron X-ray imaging) are mitigated by multiple degassing steps. The sample preparation is also compatible with magnetic resonance imaging, computed tomography and histological observation. The sample preparation and mounting require 24-36 d for a large organ such as a whole human brain or heart. The preparation time varies depending on the composition, size and fragility of the tissue. Use of the protocol enables scanning of intact organs with a diameter of 150 mm with a local voxel size of 1 μm. The protocol requires users with expertise in handling human or animal organs, laboratory operation and X-ray imaging.
AP-2β is a new mammary epithelial differentiation marker and its expression is preferentially retained and enhanced in lobular carcinoma in situ and invasive lobular breast cancer. In normal breast ...epithelium AP-2β is expressed in a scattered subpopulation of luminal cells. So far, these cells have not been further characterized. Co-expression of AP-2β protein and luminal epithelium markers (GATA3, CK8/18), hormone receptors estrogen receptor (ER), androgen receptor (AR) and candidate stem cells markers (CK5/14, CD44) were assessed by double-immunofluorescence staining in normal mammary gland epithelium. The subpopulation of AP-2β-positive mammary epithelial cells showed an almost complete, superimposable co-expression with GATA3 and a peculiar intense, ring-like appearing immunoreactivity for CK8/18. Confocal immunofluorescence microscopy revealed an apicobasal staining for CK8/18 in AP-2β-positive cells, which was not seen in in AP-2β-negative cells. Furthermore, AP-2β-positive displayed a partial co-expression with ER and AR, but lacked expression of candidate stem cell markers CK5/14 and CD44. In summary, AP-2β is a new luminal mammary epithelial differentiation marker, which is expressed in the GATA3-positive subpopulation of luminal epithelial cells. These AP-2β-positive/GATA3-positive cells also show a peculiar CK8/18-expression which may indicate a previously unknown functionally specialized mammary epithelial cell population.
Proliferative changes seen in reactive mesothelial hyperplasia of a hydrocele sac may mimic malignant mesothelioma. There is no immunohistochemical staining that reliably separates benign from ...malignant mesothelial proliferations. However, the combined analysis of BAP1 by immunohistochemistry and CDKN2A by FISH has been reported to yield both a high specificity and sensitivity in this differential diagnosis. In addition, the evaluation of risk factors such as asbestos exposure or prior traumata may be helpful for the correct diagnosis. Exclusion of stromal invasion, which is diagnostic for malign mesothelioma, is of utmost importance. Therefore, extended histological workup is essential.
Zusammenfassung
Die Abgrenzung reaktiver Mesothelläsionen gegenüber neoplastischen Prozessen des Mesothels innerhalb einer Hydrocele testis kann diagnostische Schwierigkeiten bereiten. Die ...immunhistochemische Analyse allein ist hier häufig nicht richtungsweisend, jedoch ist für die Kombination der immunhistochemischen Bestimmung von BAP1 und der FISH-Analyse von
CDKN2A
> eine hohe Spezifität und Sensitivität bezüglich dieser Differenzialdiagnose dokumentiert. Eine weitere wichtige Hilfestellung ist die Anamnese von Risikofaktoren wie Asbestbelastung und zurückliegende Traumata. Entscheidend für die korrekte Diagnosestellung einer reaktiven Mesothelläsion ist der Ausschluss von Invasivität anhand ausgedehnter Gewebeaufarbeitung.
(1) Background: In COVID-19 survivors there is an increased prevalence of pulmonary fibrosis of which the underlying molecular mechanisms are poorly understood; (2) Methods: In this multicentric ...study, n = 12 patients who succumbed to COVID-19 due to progressive respiratory failure were assigned to an early and late group (death within ≤7 and >7 days of hospitalization, respectively) and compared to n = 11 healthy controls; mRNA and protein expression as well as biological pathway analysis were performed to gain insights into the evolution of pulmonary fibrogenesis in COVID-19; (3) Results: Median duration of hospitalization until death was 3 (IQR25-75, 3-3.75) and 14 (12.5-14) days in the early and late group, respectively. Fifty-eight out of 770 analyzed genes showed a significantly altered expression signature in COVID-19 compared to controls in a time-dependent manner. The entire study group showed an increased expression of
and
, independent of hospitalization time. In the early group there was increased activity of inflammation-related genes and pathways, while fibrosis-related genes (particularly
) and pathways dominated in the late group; (4) Conclusions: After the first week of hospitalization, there is a shift from pro-inflammatory to fibrogenic activity in severe COVID-19.
and
may serve as potential therapeutic targets in future studies.
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