A Rare Case of Symptomatic Ciliated Hepatic Foregut Cyst in a 17-Year-Old Female.

A ciliated hepatic foregut cyst (CHFC) is a very uncommon cystic hepatic lesion that arises from an embryonic remnant of the foregut epithelium. CHFC is predominantly asymptomatic and is found incidentally. However, it can show various clinical presentations such as pain and weight loss. We present the case of a 17-year-old female who came to our hospital with complaints of right subcostal pain and abdominal discomfort and was diagnosed with CHFC by biopsy. Successful laparoscopic resection was performed due to the possibility of malignant transformation. Even though the majority of the patients are asymptomatic and cysts are commonly found incidentally, when the lesion has concerning features, they may need follow-up or resection due to rare reported cases of malignant transformation.

Three-dimensional Models of the Nasopharynx for the Study of Epstein-Barr Virus Infection.

The ubiquitous and cancer-associated Epstein-Barr virus (EBV) is associated with nearly all cases of nasopharyngeal carcinoma (NPC). Nasopharyngeal tissue is comprised of both pseudostratified and stratified epithelium, which are modeled in three-dimensional (3-D) cell culture. The cellular origin of EBV-associated NPC is as yet unknown, but both latent and lytic infections are likely important for preneoplastic mechanisms and replenishing the compartmentalized viral reservoir. Conventional 2-D cultures of nasopharyngeal epithelial cells (as primary cells or immortalized cell lines) are difficult to infect with EBV and cannot mimic the tissue-specific biology of the airway epithelium, which can only be captured in 3-D models. We have shown that EBV can infect the pseudostratified epithelium in air-liquid interface (ALI) culture using primary conditionally reprogrammed cells (CRCs) derived from the nasopharynx. In this protocol, we provide a step-by-step guide for the (i) conditional reprogramming of primary nasopharyngeal cells, (ii) differentiation of CRCs into pseudostratified epithelium in ALI culture (known as pseudo-ALI), and (iii) EBV infection of pseudo-ALI cultures. Additionally, we show that nasopharyngeal CRCs can be grown as organotypic rafts and subjected to EBV infection. These nasopharyngeal-derived 3-D cell cultures can be used to study EBV latent and lytic infection in relation to cell type and donor variation, by immunostaining and single-cell RNA-sequencing methods ( Ziegler et al., 2021 ). These methods are useful for studies of EBV molecular pathogenesis, and can overcome many of the limitations associated with conventional 2-D cell cultures. Graphic abstract: Workflow of nasopharyngeal-derived conditionally reprogrammed cells grown into pseudostratified-ALI and organotypic rafts in 3-D cell culture. Created with Biorender.com.

Mechanical constraints to cell-cycle progression in a pseudostratified epithelium.

As organs and tissues approach their normal size during development or regeneration, growth slows down, and cell proliferation progressively comes to a halt. Among the various processes suggested to contribute to growth termination,1-10 mechanical feedback, perhaps via adherens junctions, has been suggested to play a role.11-14 However, since adherens junctions are only present in a narrow plane of the subapical region, other structures are likely needed to sense mechanical stresses along the apical-basal (A-B) axis, especially in a thick pseudostratified epithelium. This could be achieved by nuclei, which have been implicated in mechanotransduction in tissue culture.15 In addition, mechanical constraints imposed by nuclear crowding and spatial confinement could affect interkinetic nuclear migration (IKNM),16 which allows G2 nuclei to reach the apical surface, where they normally undergo mitosis.17-25 To explore how mechanical constraints affect IKNM, we devised an individual-based model that treats nuclei as deformable objects constrained by the cell cortex and the presence of other nuclei. The model predicts changes in the proportion of cell-cycle phases during growth, which we validate with the cell-cycle phase reporter FUCCI (Fluorescent Ubiquitination-based Cell Cycle Indicator).26 However, this model does not preclude indefinite growth, leading us to postulate that nuclei must migrate basally to access a putative basal signal required for S phase entry. With this refinement, our updated model accounts for the observed progressive slowing down of growth and explains how pseudostratified epithelia reach a stereotypical thickness upon completion of growth.

Early humoral defence: Contributing to confining COVID-19 to conducting airways?

Early airway responses to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection are of interest since they could decide whether coronavirus disease-19 (COVID-19) will proceed to life-threatening pulmonary disease stages. Here I discuss endothelial-epithelial co-operative in vivo responses producing first-line, humoral innate defence opportunities in human airways. The pseudostratified epithelium of human nasal and tracheobronchial airways are prime sites of exposure and infection by SARS-CoV-2. Just beneath the epithelium runs a profuse systemic microcirculation. Its post-capillary venules respond conspicuously to mucosal challenges with autacoids, allergens and microbes, and to mere loss of epithelium. By active venular endothelial gap formation, followed by transient yielding of epithelial junctions, non-sieved plasma macromolecules move from the microcirculation to the mucosal surface. Hence, plasma-derived protein cascade systems and antimicrobial peptides would have opportunity to operate jointly on an unperturbed mucosal lining. Similarly, a plasma-derived, dynamic gel protects sites of epithelial sloughing-regeneration. Precision for this indiscriminate humoral molecular response lies in restricted location and well-regulated duration of plasma exudation. Importantly, the endothelial responsiveness of the airway microcirculation differs distinctly from the relatively non-responsive, low-pressure pulmonary microcirculation that non-specifically, almost irreversibly, leaks plasma in life-threatening COVID-19. Observations in humans of infections with rhinovirus, coronavirus 229E, and influenza A and B support a general but individually variable early occurrence of plasma exudation in human infected nasal and tracheobronchial airways. Investigations are warranted to elucidate roles of host- and drug-induced airway plasma exudation in restriction of viral infection and, specifically, whether it contributes to variable disease responses following exposure to SARS-CoV-2.

Epstein-Barr Virus Infection of Pseudostratified Nasopharyngeal Epithelium Disrupts Epithelial Integrity.

Epstein-Barr virus (EBV) is a human oncogenic virus that causes several types of tumor, such as Burkitt's lymphoma and nasopharyngeal carcinoma (NPC). NPC tumor cells are clonal expansions of latently EBV-infected epithelial cells. However, the mechanisms by which EBV transforms the nasopharyngeal epithelium is hampered, because of the lack of good in vitro model to pursue oncogenic process. Our primary nasopharyngeal epithelial cell cultures developed pseudostratified epithelium at the air-liquid interface, which was susceptible to EBV infection. Using the highly sensitive RNA in situ hybridization technique, we detected viral infection in diverse cell types, including ciliated cells, goblet cells, and basal cells. EBV-encoded small RNA-positive cells were more frequently detected in the suprabasal layer than in the basal layer. We established the most physiologically relevant EBV infection model of nasopharyngeal epithelial cells. This model will advance our understanding of EBV pathogenesis in the development of NPC.

RYK-mediated filopodial pathfinding facilitates midgut elongation.

Between embryonic days 10.5 and 14.5, active proliferation drives rapid elongation of the murine midgut epithelial tube. Within this pseudostratified epithelium, nuclei synthesize DNA near the basal surface and move apically to divide. After mitosis, the majority of daughter cells extend a long, basally oriented filopodial protrusion, building a de novo path along which their nuclei can return to the basal side. WNT5A, which is secreted by surrounding mesenchymal cells, acts as a guidance cue to orchestrate this epithelial pathfinding behavior, but how this signal is received by epithelial cells is unknown. Here, we have investigated two known WNT5A receptors: ROR2 and RYK. We found that epithelial ROR2 is dispensable for midgut elongation. However, loss of Ryk phenocopies the Wnt5a-/- phenotype, perturbing post-mitotic pathfinding and leading to apoptosis. These studies reveal that the ligand-receptor pair WNT5A-RYK acts as a navigation system to instruct filopodial pathfinding, a process that is crucial for continuous cell cycling to fuel rapid midgut elongation.

Hedgehog Signaling Regulates Epithelial Morphogenesis to Position the Ventral Embryonic Midline.

Despite much progress toward understanding how epithelial morphogenesis is shaped by intra-epithelial processes including contractility, polarity, and adhesion, much less is known regarding how such cellular processes are coordinated by extra-epithelial signaling. During embryogenesis, the coelomic epithelia on the two sides of the chick embryo undergo symmetrical lengthening and thinning, converging medially to generate and position the dorsal mesentery (DM) in the embryonic midline. We find that Hedgehog signaling, acting through downstream effectors Sec5 (ExoC2), an exocyst complex component, and RhoU (Wrch-1), a small GTPase, regulates coelomic epithelium morphogenesis to guide DM midline positioning. These effects are accompanied by changes in epithelial cell-cell alignment and N-cadherin and laminin distribution, suggesting Hedgehog regulation of cell organization within the coelomic epithelium. These results indicate a role for Hedgehog signaling in regulating epithelial morphology and provide an example of how transcellular signaling can modulate specific cellular processes to shape tissue morphogenesis.

Tissue Mechanics Regulate Mitotic Nuclear Dynamics during Epithelial Development.

Cell divisions are essential for tissue growth. In pseudostratified epithelia, where nuclei are staggered across the tissue, each nucleus migrates apically before undergoing mitosis. Successful apical nuclear migration is critical for planar-orientated cell divisions in densely packed epithelia. Most previous investigations have focused on the local cellular mechanisms controlling nuclear migration. Inter-species and inter-organ comparisons of different pseudostratified epithelia suggest global tissue architecture may influence nuclear dynamics, but the underlying mechanisms remain elusive. Here, we use the developing Drosophila wing disc to systematically investigate, in a single epithelial type, how changes in tissue architecture during growth influence mitotic nuclear migration. We observe distinct nuclear dynamics at discrete developmental stages, as epithelial morphology changes. We use genetic and physical perturbations to show a direct effect of cell density on mitotic nuclear positioning. We find Rho kinase and Diaphanous, which facilitate mitotic cell rounding in confined cell conditions, are essential for efficient apical nuclear movement. Perturbation of Diaphanous causes increasing defects in apical nuclear migration as the tissue grows and cell density increases, and these defects can be reversed by acute physical reduction of cell density. Our findings reveal how the mechanical environment imposed on cells within a tissue alters the molecular and cellular mechanisms adopted by single cells for mitosis.

Gastric duplication cyst lined by pseudostratified columnar ciliated epithelium masquerading as a pancreatic mucinous cystic neoplasm: a case report and literature review.

Gastric duplication cyst (GDC) with a pseudostratified columnar ciliated epithelial (PCCE) is a congenital rare cystic neoplasm, which is often difficult to distinguish from other entities by imaging techniques, and as a consequence it may be wrongly overtreated. We herein report a case of a 52-year-old female incidentally found to have an abdominal mass by ultrasonography and computed tomography. Additionally, endoscopic ultrasonography and fluid analysis were consistent with a pancreatic mucinous cystic neoplasm with a markedly elevated fluid amylase, carcinoembryonic antigen, and carbohydrate antigen 19-9. Then, laparoscopic resection of the cyst originating from the stomach and wedge gastrectomy were performed. Final pathology revealed a GDC with PCCE. In addition, we also performed a literature review of 31 reports of GDC with PCCE. Although rare, GDC lined by PCCE should be included in the differential diagnosis of pancreatic cystic neoplasms or a gastric wall mass.

Non-woven bilayered biodegradable chitosan-gelatin-polylactide scaffold for bioengineering of tracheal epithelium.

OBJECTIVES: The conversion of tissue engineering into a routine clinical tool cannot be achieved without a deep understanding of the interaction between cells and scaffolds during the process of tissue formation in an artificial environment. Here, we have investigated the cultivation conditions and structural features of the biodegradable non-woven material in order to obtain a well-differentiated human airway epithelium. MATERIALS AND METHODS: The bilayered scaffold was fabricated by electrospinning technology. The efficiency of the scaffold has been evaluated using MTT cell proliferation assay, histology, immunofluorescence and electron microscopy. RESULTS: With the use of a copolymer of chitosan-gelatin-poly-l-lactide, a bilayered non-woven scaffold was generated and characterized. The optimal structural parameters of both layers for cell proliferation and differentiation were determined. The basal airway epithelial cells differentiated into ciliary and goblet cells and formed pseudostratified epithelial layer on the surface of the scaffold. In addition, keratinocytes formed a skin equivalent when seeded on the same scaffold. A comparative analysis of growth and differentiation for both types of epithelium was performed. CONCLUSIONS: The structural parameters of nanofibres should be selected experimentally depending on polymer composition. The major challenges on the way to obtain the well-differentiated equivalent of respiratory epithelium on non-woven scaffold include the following: the balance between scaffold permeability and thickness, proper combination of synthetic and natural components, and culture conditions sufficient for co-culturing of airway epithelial cells and fibroblasts. For generation of skin equivalent, the lack of diffusion is not so critical as for pseudostratified airway epithelium.

Measurement of Mitotic Spindle Angle and Mitotic Cell Distance in Fixed Tissue of Drosophila Larval Brains.

The positioning and the cleavage plane orientation of mitotic cells in pseudostratified epithelia (PSE) must be tightly regulated since failures in any of these processes might have fatal consequences during development. Here we present a simple method to determine the spindle orientation as well as the positioning of neuroepithelial mitotic cells within the Outer Proliferation Center (OPC) of Drosophila larval brains.

Expansion of Airway Basal Cells and Generation of Polarized Epithelium.

Airway basal stem cells are the progenitor cells within the airway that exhibit the capacity to self-renew and give rise to multiple types of differentiated airway epithelial cells. This stem cell-derived epithelium displays organized architecture with functional attributes of the airway mucosa. A protocol has been developed to culture and expand human airway basal stem cells while preserving their stem cell properties and capacity for subsequent mucociliary differentiation. This achievement presents a previously unrealized opportunity to maintain a durable supply of progenitor cells derived from healthy donors to differentiate into human primary airway epithelium for cellular and molecular-based studies. Further, basal stem cells can be harvested from patients with a specific airway disease, such as cystic fibrosis, enabling investigation of potentially altered behavior of disease-specific cells in the appropriate context of the airway mucosa. Here we describe, in detail, a protocol for the serial expansion of airway basal stem cells to enable the generation of nearly unlimited airway basal cells that can be stored and readily available for subsequent culturing and differentiation. In addition, we describe culturing and differentiation of airway basal stem cells on permeable transwell filters at air-liquid interface to create functional mucociliary pseudostratified polarized airway epithelial mucosa.

Radial WNT5A-Guided Post-mitotic Filopodial Pathfinding Is Critical for Midgut Tube Elongation.

The early midgut undergoes intensive elongation, but the underlying cellular and molecular mechanisms are unknown. The early midgut epithelium is pseudostratified, and its nuclei travel between apical and basal surfaces in concert with cell cycle. Using 3D confocal imaging and 2D live imaging, we profiled behaviors of individual dividing cells. As nuclei migrate apically for mitosis, cells maintain a basal process (BP), which splits but is inherited by only one daughter. After mitosis, some daughters directly use the inherited BP as a "conduit" to transport the nucleus basally, while >50% of daughters generate a new basal filopodium and use it as a path to return the nucleus. Post-mitotic filopodial "pathfinding" is guided by mesenchymal WNT5A. Without WNT5A, some cells fail to tether basally and undergo apoptosis, leading to a shortened midgut. Thus, these studies reveal previously unrecognized strategies for efficient post-mitotic nuclear trafficking, which is critical for early midgut elongation.

Differentiating cells mechanically limit the interkinetic nuclear migration of progenitor cells to secure apical cytogenesis.

Many proliferative epithelia are pseudostratified because of cell cycle-dependent interkinetic nuclear migration (IKNM, basal during G1 and apical during G2). Although most epithelia, including early embryonic neuroepithelia (≤100 µm thick), undergo IKNM over the entire apicobasal extent, more apicobasally elongated (300 µm) neural progenitor cells (radial glial cells) in the mid-embryonic mouse cerebral wall move their nuclei only within its apical (100 µm) compartment, leaving the remaining basal region nucleus-free (fiber-like). How this IKNM range [i.e. the thickness of a pseudostratified ventricular zone (VZ)] is determined remains unknown. Here, we report external fencing of IKNM and the VZ by differentiating cells. When a tight stack of multipolar cells immediately basal to the VZ was 'drilled' via acute neuron-directed expression of diphtheria toxin, IKNM of apicobasally connected progenitor cells continued further towards the basal region of the cell (200 µm). The unfencing-induced basally overshot nuclei stay in S phase for too long and do not move apically, suggesting that external limitation of IKNM is necessary for progenitors to undergo normal cytogenetic behaviors. Thus, physical collaboration between progenitors and differentiating cells, including neurons, underlies brain development.

Molecular components and polarity of radial glial cells during cerebral cortex development.

Originating from ectodermal epithelium, radial glial cells (RGCs) retain apico-basolateral polarity and comprise a pseudostratified epithelial layer in the developing cerebral cortex. The apical endfeet of the RGCs faces the fluid-filled ventricles, while the basal processes extend across the entire cortical span towards the pial surface. RGC functions are largely dependent on this polarized structure and the molecular components that define it. In this review, we will dissect existing molecular evidence on RGC polarity establishment and during cerebral cortex development and provide our perspective on the remaining key questions.

Large bronchogenic cyst of stomach: A case report.

INTRODUCTION: Bronchogenic cysts are congenital cysts arising as an abnormal budding from primitive tracheobronchial tree. They are lined by pseudostratified columnar or cuboidal ciliated epithelium and contain smooth muscle fibers, submucosal bronchial glands and/or cartilage. They are most frequently located in the mediastinum or the lung parenchyma. Intramural occurrence of bronchogenic cyst in the gastric wall is very rare. PRESENTATION OF CASE: We present a case of 65-year-old lady with a 7×8cm lesion in the gastric cardia suspicious of gastrointestinal stromal tumor. Because of the large size, total gastrectomy with Roux-en-Y esophagojejunal anastomosis was performed. The postoperative course was uneventful. Histopathological examination revealed a sub-mucosal cyst lined by PCCE with presence of smooth muscle fibers and focal mucous glands. Final diagnosis of bronchogenic cyst was made. On the last follow up at one year, she was symptom free. DISCUSSION: On extensive Medline/Pubmed search, only 38 cases of gastric bronchogenic cysts were found to be reported till date. They are typically located in the posterior gastric wall close to the gastric cardia. On radiological imaging, they appear as well defined intramural cystic lesion without any characteristic features. Surgical resection is considered in symptomatic cases or in case of diagnostic dilemma. CONCLUSION: Gastric bronchogenic cysts often mimic gastrointestinal stromal tumor on preoperative imaging. They should be included in the differential diagnosis while dealing with an intramural gastric lesion close to the cardia or gastroesophageal junction.

Ciliated hepatic foregut cyst: report of three cases and review of imaging features.

Ciliated hepatic foregut cysts (CHFCs) are rare cystic lesions which are most commonly asymptomatic. They can be clinically important as they may, on rare occasions, undergo malignant transformation or cause mass effect if significantly enlarged. Three cases of CHFCs are presented in this article and their imaging features are reviewed.

Heterogeneity, Cell Biology and Tissue Mechanics of Pseudostratified Epithelia: Coordination of Cell Divisions and Growth in Tightly Packed Tissues.

Pseudostratified epithelia (PSE) are tightly packed proliferative tissues that are important precursors of the development of diverse organs in a plethora of species, invertebrate and vertebrate. PSE consist of elongated epithelial cells that are attached to the apical and basal side of the tissue. The nuclei of these cells undergo interkinetic nuclear migration (IKNM) which leads to all mitotic events taking place at the apical surface of the epithelium. In this review, we discuss the intricacies of proliferation in PSE, considering cell biological, as well as the physical aspects. First, we summarize the principles governing the invariability of apical nuclear migration and apical cell division as well as the importance of apical mitoses for tissue proliferation. Then, we focus on the mechanical and structural features of these tissues. Here, we discuss how the overall architecture of pseudostratified tissues changes with increased cell packing. Lastly, we consider possible mechanical cues resulting from these changes and their potential influence on cell proliferation.

Electron microscope based X-ray microanalysis on bioaccumulation of heavy metals and neural degeneration in mudskipper [Pseudapocryptes lanceolatus].

The bioaccumulation of heavy metals and its probable cytological consequences in ciliated olfactory sensory receptor neuron (OSN) of two different groups of Pseudapocryptes lanceolatus has been studied using X-ray microanalysis in transmission electron microscopy (TEM-EDX) [i.e., Group I, collected near Kanchrapara (22.56°N 88.26°E) and Group II, collected near Tribeni (22.99°N 88.40°E) of West Bengal, India]. The ciliated OSN is a bipolar neuron and possesses a prolonged dendron with four to six cilia at the olfactory knob, perikaryon, and axon. Excess accumulation of copper (94.50%) and iron (83.81%) was noted under TEM-EDX in the cytoplasm of the olfactory knob as well as nucleoplasm of ciliated OSNs in P. lanceolatus (Group II). The degenerating ciliated OSNs show distinct features of lysis of the plasma membrane at the olfactory knob, disintegration of cytoskeletal structures in perinuclear cytoplasm and axoplasm, and fragmented chromatin fibers with granules (diameter, 20-30 nm) in the nucleoplasm. Crowding of acetylcholinesterase-positive vesicles (diameter:, 30-40 nm) at the terminal part of the axoplasm was related to accumulation of heavy metals in degenerating ciliated OSNs of P. lanceolatus (Group II). The recorded concentrations of heavy metals in the same organ among different groups of P. lanceolatus in varying geographical areas indicates the stress of concerned environmental health. This ultrastructural interpretation on the fish ciliated OSN is a prerequisite for monitoring environmental health as well as metallobiology of several neurodegenerative disorders in fish caused by bioaccumulation of heavy metals.