Well-differentiated Epithelium Research Articles

Fibroblasts and polymer composition are essential for bioengineering of airway epithelium on nonwoven scaffolds

To make tissue engineering a truly effective tool, it is necessary to understand how the patterns of specific tissue development are modulated by and depend on the artificial environment. Even the most advanced approaches still do not fully meet the requirements of practical engineering of tracheobronchial epithelium. This study aimed to test the ability of the synthetic and natural nonwoven scaffolds to support the formation of morphological sound airway epithelium including the basement membrane (BM). We also sought to identify the potential role of fibroblasts in this process. Our results showed that nonwoven scaffolds are generally suitable for producing well-differentiated tracheobronchial epithelium (with cilia and goblet cells), while the structure and functionality of the equivalents appeared to be highly dependent on the composition of the scaffolds. Unlike natural scaffolds, synthetic ones supported the formation of the epithelium only when epithelial cells were cocultured with fibroblasts. Fibroblasts also appeared to be obligatory for basal lamina formation, regardless of the type of the nonwoven material used. However, even in the presence of fibroblasts, the synthetic scaffolds were unable to support the formation of the epithelium and of the BM (in particular, basal lamina) as effectively as the natural scaffolds did.

Surfactant protein D prevents mucin overproduction in airway goblet cells via SIRPα

Mucin overproduction is a common feature of chronic airway diseases such as asthma and chronic obstructive pulmonary disease (COPD), and exacerbates their underlying respiratory condition. Surfactant protein D (SP-D) protects against airway diseases through modulation of immune reactions, but whether it also exerts direct effects on airway epithelial cells has remained unclear. Therefore, we sought to investigate the inhibitory role of SP-D on mucin production in airway epithelial cells. We prepared air–liquid interface (ALI) cultures of human primary bronchial epithelial cells (HBECs), which recapitulated a well-differentiated human airway epithelium. Benzo(a)pyrene (BaP), a key toxicant in cigarette smoke, induced mucin 5AC (MUC5AC) production in ALI-cultured HBECs, airway secretory cell lines, and airway epithelia of mice. Then, the protective effects of SP-D against the BaP-induced mucin overproduction were examined. BaP increased MUC5AC production in ALI cultures of HBECs, and this effect was attenuated by SP-D. SP-D also suppressed the BaP-induced phosphorylation of extracellular signal-regulated kinase (ERK) and MUC5AC expression in NCI-H292 goblet-like cells, but not in NCI-H441 club-like cells. Signal regulatory protein α (SIRPα) was found to be expressed in HBECs and NCI-H292 cells but absent in NCI-H441 cells. In NCI-H292 cells, SP-D activated SH2 domain-containing tyrosine phosphatase-1 (SHP-1), downstream of SIRPα, and knockdown of SIRPα abolished the suppressive effects of SP-D on BaP-induced ERK phosphorylation and MUC5AC production. Consistent with these in vitro findings, intratracheal instillation of SP-D prevented the BaP-induced phosphorylation of ERK and Muc5ac expression in airway epithelial cells in a mouse model. SP-D acts directly on airway epithelial cells to inhibit mucin secretion through ligation of SIRPα and SHP-1-mediated dephosphorylation of ERK. Targeting of SIRPα is therefore a potential new therapeutic approach to suppression of mucin hypersecretion in chronic airway diseases such as COPD and asthma.

Severe pneumonia and pathogenic damage in human airway epithelium caused by Coxsackievirus B4

ABSTRACT Coxsackievirus B4 (CVB4) has one of the highest proportions of fatal outcomes of other enterovirus serotypes. However, the pathogenesis of severe respiratory disease caused by CVB4 infection remains unclear. In this study, 3 of 42 (7.2%, GZ-R6, GZ-R7 and GZ-R8) patients with severe pneumonia tested positive for CVB4 infection in southern China. Three full-length genomes of pneumonia-derived CVB4 were sequenced and annotated for the first time, showing their high nucleotide similarity and clustering within genotype V. To analyze the pathogenic damage caused by CVB4 in the lungs, a well-differentiated human airway epithelium (HAE) was established and infected with the pneumonia-derived CVB4 isolate GZ-R6. The outcome was compared with that of a severe hand-foot-mouth disease (HFMD)-derived CVB4 strain GZ-HFM01. Compared with HFMD-derived CVB4, pneumonia-derived CVB4 caused more intense and rapid disruption of HAE polarity, leading to tight-junction barrier disruption, loss of cilia, and airway epithelial cell hypertrophy. More pneumonia-derived CVB4 were released from the basolateral side of the HAE than HFMD-derived CVB4. Of the 18 cytokines tested, only IL-6 and IL-1b secretion significantly increased on bilateral sides of HAE during the early stage of pneumonia-derived CVB4 infection, while multiple cytokine secretions significantly increased in HFMD-derived CVB4-infected HAE. HFMD-derived CVB4 exhibited stronger neurovirulence in the human neuroblastoma cells SH-SY5Y than pneumonia-derived CVB4, which is consistent with the clinical manifestations of patients infected with these two viruses. This study has increased the depth of our knowledge of severe pneumonia infection caused by CVB4 and will benefit its prevention and treatment.

Down syndrome is associated with altered frequency and functioning of tracheal multiciliated cells, and response to influenza virus infection

Individuals with Down syndrome (DS) clinically manifest severe respiratory illnesses; however, there is a paucity of data on how DS influences homeostatic physiology of lung airway, and its reactive responses to pulmonary pathogens. We generated well-differentiated ciliated airway epithelia using tracheas from wild-type and Dp(16)1/Yey mice invitro, and discovered that Dp(16)1/Yey epithelia have significantly lower abundance of ciliated cells, an altered ciliary beating profile, and reduced mucociliary transport. Interestingly, both sets of differentiated epithelia released similar quantities of viral particles after infection with influenza A virus (IAV). However, RNA-sequencing and proteomic analyses revealed an immune hyperreactive phenotype particularly for monocyte-recruiting chemokines in Dp(16)1/Yey epithelia. Importantly, when we challenged mice invivo with IAV, we observed immune hyper-responsiveness in Dp(16)1/Yey mice, evidenced by higher quantities of lung airway infiltrated monocytes, and elevated levels of pro-inflammatory cytokines in bronchoalveolar lavage fluid. Our findings illuminate mechanisms underlying DS-mediated pathophysiological changes in airway epithelium.

Redox Homeostasis in Well-differentiated Primary Human Nasal Epithelial Cells.

Oxidative stress (OS) in the airway epithelium is associated with inflammation, cell damage, and mitochondrial dysfunction that may initiate or worsen respiratory disease. Redox regulation maintains the equilibrium of pro-oxidant/antioxidant reactions but can be disturbed by environmental exposures. The mechanism(s) underlying the induction and impact of OS on airway epithelium and how these influences on respiratory disease is poorly understood. The aim of this study was to develop a stress response model in primary human nasal epithelial cells (NECs) grown at the air-liquid interface (ALI) into a well-differentiated epithelium and to use this model to investigate the mechanisms underlying OS. Hydrogen peroxide (H2O2) was used to induce acute OS and the responses were measured with trans epithelial electrical resistance (TEER), membrane permeability, cell death (LDH release), mitochondrial reactive oxygen species (mtROS) generation, redox status (GSH/GSSG ratio), cellular ATP, and signaling pathways (SIRT1, FOXO3, p53, p21, PINK1, PARKIN, NRF2). Following 25 mM (sensitive) or 50mM (resistant) H2O2 exposure, cell integrity decreased (p<0.05), GSH/GSSG ratio reduced (p<0.05), and ATP production declined by 83% (p<0.05) in the sensitive and 55% (p<0.05) in the resistant group; mtROS production increased 3.4-fold (p<0.001). Significant inter-individual differences between healthy humans with regards to susceptibility to OS, and differential activation of various pathways (FOXO3, PARKIN) were observed. These intra-individual differences in susceptibility to OS may be attributed to resistant individuals having more mitochondria or greater mitochondrial function.

Resveratrol and Astaxanthin Protect Primary Human Nasal Epithelial Cells Cultured at an Air-liquid Interface from an Acute Oxidant Exposure.

Oxidative stress (OS) in the airway epithelium is associated with cell damage, inflammation, and mitochondrial dysfunction that may initiate or worsen respiratory disease. However, it is unclear whether exogenous antioxidants can provide protection to the airway epithelium from OS. Resveratrol and astaxanthin are nutritional compounds that have shown diverse benefits including protection against OS and inflammation in various situations. The aim of this study was to examine the utility of pre-treatment with resveratrol and astaxanthin to prevent the negative effects of oxidant exposure and restore redox homeostasis in a well-differentiated epithelium grown from primary human nasal epithelial cells (NECs) at the air-liquid interface. Fully differentiated NECs were pretreated with the antioxidants for 24 hours and the cultured epithelia was subsequently exposed to hydrogen peroxide (H2O2) for 1 hour to induce an acute OS. Responses measured included mitochondrial reactive oxygen species (mtROS) generation, redox status (GSH/GSSG ratio), cellular ATP, and signaling pathways (SIRT1, FOXO3, p21, PINK1, PARKIN, NRF2). Following H2O2 exposure, mtROS production increased by 4-fold compared with control (p<0.01) and pre-treatment with resveratrol or astaxanthin reduced this by 50% (p<0.05). H2O2 exposure reduced GSH/GSSG ratio and this decline was prevented by antioxidants pre-treatment. H2O2 exposure caused 2.5-fold increase in p21 mRNA expression compared with control (p<0.05), while a slight decrease in p21 mRNA expression was observed when cells were pre-treated with resveratrol or astaxanthin. Our results demonstrate that antioxidants, resveratrol, and astaxanthin were able to protect cells from an acute OS. These agents show promise that encourages further research.

Scaffold-Free Tracheal Engineering via a Modular Strategy Based on Cartilage and Epithelium Sheets.

Tracheal defects lead to devastating problems, and practical clinical substitutes that have complex functional structures and can avoid adverse influences from exogenous bioscaffolds are lacking. Herein, a modular strategy for scaffold-free tracheal engineering is developed. A cartilage sheet (Cart-S) prepared by high-density culture is laminated and reshaped to construct a cartilage tube as the main load-bearing structure in which the chondrocytes exhibit a stable phenotype and secreted considerable cartilage-specific matrix, presenting a native-like grid arrangement. To further build a tracheal epithelial barrier, a temperature-sensitive technique is used to construct the monolayer epithelium sheet (Epi-S), in which the airway epithelial cells present integrated tight junctions, good transepithelial electrical resistance, and favorable ciliary differentiation capability. Epi-S can be integrally transferred to inner wall of cartilage tube, forming a scaffold-free complex tracheal substitute (SC-trachea). Interestingly, when Epi-S is attached to the cartilage surface, epithelium-specific gene expression is significantly enhanced. SC-trachea establishes abundant blood supply via heterotopic vascularization and then is pedicle transplanted for tracheal reconstruction, achieving 83.3% survival outcomes in rabbit models. Notably, the scaffold-free engineered trachea simultaneously satisfies sufficient mechanical properties and barrier function due to its matrix-rich cartilage structure and well-differentiated ciliated epithelium, demonstrating great clinical potential for long-segmental tracheal reconstruction.

Immunoresponsive microbiota-gut-on-chip reproduces barrier dysfunction, stromal reshaping and probiotics translocation under inflammation

Here, we propose an immune-responsive human Microbiota-Intestine axis on-chip as a platform able to reproduce the architecture and vertical topography of the microbiota with a complex extracellular microenvironment consisting of a responsive extra cellular matrix (ECM) and a plethora of immune-modulatory mediators released from different cell populations such as epithelial, stromal, blood and microbial species in homeostatic and inflamed conditions. Firstly, we developed a three-dimensional human intestine model (3D-hI), represented by an instructive and histologically competent ECM and a well-differentiated epithelium with mucus-covered microvilli. Then, we replicated the microenvironmental anaerobic condition of human intestinal lumen by fabricating a custom-made microbiota chamber (MC) on the apical side of the Microbiota-human Intestine on chip (MihI-oC), establishing the physiological oxygen gradient occurring along the thickness of human small intestine from the serosal to the luminal side. The complexity of the intestinal extracellular microenvironment was improved by integrating cells populations that are directly involved in the inflammatory response such as peripheral blood mononuclear cells (PBMCs) and two species of the intestinal commensal microbiota (Lactobacillus rhamnosus and Bifidobacterium longum). We found that lipopolysaccharide (LPS)-induced inflammation elicits microbiota's geographical change and induce Bifidobacterium longum iper-proliferation, highlighting a role of such probiotic in anti-inflammatory process. Moreover, we proved, for the first time, the indirect role of the microbiota on stromal reshaping in immune-responsive MihI-oC in terms of collagen fibers orientation and ECM remodeling, and demonstrated the role of microbiota in alleviating gastrointestinal, immunological and infectious diseases by analyzing the release of key immune-mediators after inflammatory stimulus (reactive oxygen species (ROS), pro- and anti-inflammatory cytokines).

Neutrophil Extracellular Traps Do Not Induce Injury and Inflammation in Well-Differentiated RSV-Infected Airway Epithelium.

Respiratory syncytial virus (RSV) lower respiratory tract infection (LRTI) causes a major burden of disease. The host response in RSV-LRTI is characterized by airway epithelial injury, inflammation and neutrophil influx, with the formation of neutrophil extracellular traps (NETs). However, the precise role of NETs in the pathophysiology of RSV-LRTI remains to be elucidated. Here, we used well-differentiated human airway epithelial cultures (HAE) of a pediatric and adult donor to study whether NETs cause airway epithelial injury and inflammation in the setting of RSV infection. The exposure of uninfected and RSV-infected HAE cultures to NETs, as produced by stimulation of neutrophils by a low dose of phorbol 12-myristate 13-acetate (PMA), did not induce or aggravate cell injury or inflammation. RSV infection of HAE cultures caused release of pro-inflammatory cytokines such as IL-6 and RANTES in both adult and pediatric cultures, but the differential gene expression for regulated cell death differed between culture donors. In this in vitro airway epithelial model, NETs in the setting of RSV infection did not cause or aggravate epithelial injury or inflammation.

Functional and Transcriptional Adaptations of Blood Monocytes Recruited to the Cystic Fibrosis Airway Microenvironment In Vitro.

Cystic fibrosis (CF) lung disease is dominated by the recruitment of myeloid cells (neutrophils and monocytes) from the blood which fail to clear the lung of colonizing microbes. In prior in vitro studies, we showed that blood neutrophils migrated through the well-differentiated lung epithelium into the CF airway fluid supernatant (ASN) mimic the dysfunction of CF airway neutrophils in vivo, including decreased bactericidal activity despite an increased metabolism. Here, we hypothesized that, in a similar manner to neutrophils, blood monocytes undergo significant adaptations upon recruitment to CFASN. To test this hypothesis, primary human blood monocytes were transmigrated in our in vitro model into the ASN from healthy control (HC) or CF subjects to mimic in vivo recruitment to normal or CF airways, respectively. Surface phenotype, metabolic and bacterial killing activities, and transcriptomic profile by RNA sequencing were quantified post-transmigration. Unlike neutrophils, monocytes were not metabolically activated, nor did they show broad differences in activation and scavenger receptor expression upon recruitment to the CFASN compared to HCASN. However, monocytes recruited to CFASN showed decreased bactericidal activity. RNASeq analysis showed strong effects of transmigration on monocyte RNA profile, with differences between CFASN and HCASN conditions, notably in immune signaling, including lower expression in the former of the antimicrobial factor ISG15, defensin-like chemokine CXCL11, and nitric oxide-producing enzyme NOS3. While monocytes undergo qualitatively different adaptations from those seen in neutrophils upon recruitment to the CF airway microenvironment, their bactericidal activity is also dysregulated, which could explain why they also fail to protect CF airways from infection.

Human Bocavirus 1 Infection of Well-Differentiated Human Airway Epithelium.

Human bocavirus 1 (HBoV1) is a small DNA virus that belongs to the Bocaparvovirus genus of the Parvoviridae family. HBoV1 is a common respiratory pathogen that causes mild to life-threatening acute respiratory tract infections in children and immunocompromised individuals, infecting both the upper and lower respiratory tracts. HBoV1 infection causes death of airway epithelial cells, resulting in airway injury and inflammation. In vitro, HBoV1 only infects well-differentiated (polarized) human airway epithelium cultured at an air-liquid interface (HAE-ALI), but not any dividing human cells. A full-length HBoV1 genome of 5543 nucleotides has been cloned from DNA extracted from a human nasopharyngeal swab into a plasmid called HBoV1 infectious clone pIHBoV1. Transfection of pIHBoV1 replicates efficiently in human embryonic kidney 293 (HEK293) cells and produces virions that are highly infectious. This article describes protocols for production of HBoV1 in HEK293 cells, generation of HAE-ALI cultures, and infection with HBoV1 in HAE-ALI. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Human bocavirus 1 production in HEK293 cells Support Protocol 1: HEK293 cell culture and transfection Support Protocol 2: Quantification of human bocavirus 1 using real-time quantitative PCR Basic Protocol 2: Differentiation of human airway cells at an air-liquid interface Support Protocol 3: Expansion of human airway epithelial cell line CuFi-8 Support Protocol 4: Expansion of human airway basal cells Support Protocol 5: Coating of plastic dishes and permeable membranes of inserts Support Protocol 6: Transepithelial electrical resistance measurement Basic Protocol 3: Human bocavirus 1 infection in human airway epithelium cultured at an air-liquid interface Support Protocol 7: Isolation of infected human airway epithelium cells from inserts Basic Protocol 4: Transduction of airway basal cells with lentiviral vector.

The Self-assembly Approach as a Tool for the Tissue Engineering of a Bi-lamellar Human Cornea.

Tissue engineering is a flourishing field of regenerative medicine that allows the reconstruction of various tissues of our body, including the cornea. In addition to addressing the growing need for organ transplants, such tissue-engineered substitutes may also serve as good in vitro models for fundamental and preclinical studies. Recent progress in the field of corneal tissue engineering has led to the development of new technologies allowing the reconstruction of a human bi-lamellar cornea. One unique feature of this model is the complete absence of exogenous material. Indeed, these human corneal equivalents are exclusively composed of untransformed human corneal fibroblasts (hCFs) entangled in their own extracellular matrix, as well as untransformed human corneal epithelial cells (hCECs), both of which isolated from donor corneas. The reconstructed human bi-lamellar cornea thereby exhibits a well-organized stroma as well as a well-differentiated epithelium. This chapter describes the methods used for the isolation and culture of hCFs, the production and assembly of hCFs stromal sheets, the seeding of hCECs, and the maturation of the tissue-engineered cornea.

Role of Cytokeratin-7 in the pathogenesis of odontogenic cysts - an immunohistochemical study.

IntroductionOdontogenic cysts are distinct entities and quite a common occurrence in the jaw bones. These are individual lesions which arise from the same odontogenic apparatus but with varying pathogenesis. Cytokeratins are integral components in tooth development and are expressed across the odontogenic tissues in physiological and pathological states.AimTo elucidate the role of cytokeratin-7 in the pathogenesis of odontogenic cysts by immunohistochemistry.MethodCytokeratin-7 (CK-7) was assessed in 39 cases of odontogenic lesions retrieved from the archival files which included 15 cases of dentigerous cysts (DC), 12 cases of odontogenic keratocysts (OKC) and 12 cases of radicular cysts (RC) and also 8 cases of control specimens.Statistical analysisResults obtained were statistically analyzed using chi-square test to assess the association between different odontogenic cysts used in this study and Cytokeration-7 staining. The difference was considered to be of statistical significance if the p value was ≤ 0.05.ResultsCK7 expression was maximum in dentigerous cycts (66.66%) followed by radicular cysts (41.66%) and odontogenic keratocysts (16.6%). On evaluation of staining and expression pattern, highest positivity is shown in dentigerous cysts and the positivity is seen in suprabasal (60%) and superficial layers (40%) whereas radicular cysts and odontogenic keratocysts showed positivity in superficial and spinous layers.ConclusionCytokeratin-7 expression correlates with the degree of differentiation of the epithelium. So the cysts with a well-differentiated epithelium (RC and DC) express CK-7, while the cysts with a less well-differentiated epithelium (OKC) show slight positivity. Thus it can be useful to differentiate OKC from DC and RC.

An ALI culture well with well-differentiated respiratory epithelium demonstrating coordinated ciliary activity and mucociliary clearance

An ALI culture well with well-differentiated respiratory epithelium demonstrating coordinated ciliary activity and mucociliary clearance

An ALI culture well exhibiting well-differentiated respiratory epithelium with ciliary activity

An ALI culture well exhibiting well-differentiated respiratory epithelium with ciliary activity

A Comprehensive RNA-seq Analysis of Human Bocavirus 1 Transcripts in Infected Human Airway Epithelium.

Human bocavirus 1 (HBoV1) infects well-differentiated (polarized) human airway epithelium (HAE) cultured at an air-liquid interface (ALI). In the present study, we applied next-generation RNA sequencing to investigate the genome-wide transcription profile of HBoV1, including viral mRNA and small RNA transcripts, in HBoV1-infected HAE cells. We identified novel transcription start and termination sites and confirmed the previously identified splicing events. Importantly, an additional proximal polyadenylation site (pA)p2 and a new distal polyadenylation site (pA)dREH lying on the right-hand hairpin (REH) of the HBoV1 genome were identified in processing viral pre-mRNA. Of note, all viral nonstructural proteins-encoding mRNA transcripts use both the proximal polyadenylation sites [(pA)p1 and (pA)p2] and distal polyadenylation sites [(pA)d1 and (pA)dREH] for termination. However, capsid proteins-encoding transcripts only use the distal polyadenylation sites. While the (pA)p1 and (pA)p2 sites were utilized at roughly equal efficiency for proximal polyadenylation of HBoV1 mRNA transcripts, the (pA)d1 site was more preferred for distal polyadenylation. Additionally, small RNA-seq analysis confirmed there is only one viral noncoding RNA (BocaSR) transcribed from nt 5199–5340 of the HBoV1 genome. Thus, our study provides a systematic and unbiased transcription profile, including both mRNA and small RNA transcripts, of HBoV1 in HBoV1-infected HAE-ALI cultures.

The wound healing capacity of undifferentiated and differentiated airway epithelial cells in vitro

IntroductionCongenital or acquired tracheal lesions alter airway epithelial structure and can lead to long-segment tracheal defects. Tissue engineered tracheal grafts (TETG) have the potential to cure such defects; however, clinical applications have been plagued with numerous complications including delayed graft epithelialization. The knowledge that epithelial cells migrate from native tissue to the TETG raises the possibility that TETG performance can be improved by increasing the rate of epithelialization. ObjectivesWe developed a model that can be used quantify epithelial migration in clinically-relevant conditions. MethodsExisting histological analyses determined the differentiation status of the normal and injured human tracheal epithelium and were used to identify in vitro culture conditions that mimic these parameters. The classical scratch assay was adapted to permit analysis of migratory velocity as a function of differentiation status. Migration of undifferentiated (UD), partially-differentiated (PD), and well-differentiated (WD) epithelia was quantified. ResultsThe normal and injured epithelium can be modeled using human cells that are cultured using a modified air-liquid-interface culture system. PD cell cultures are similar to the remodeled epithelium; whereas; WD cultures are similar to the normal epithelium. Preliminary results indicate that PD cells migrate more rapidly than WD cells and that PD and WD cells migrate more rapidly than UD cells. ConclusionPending verification of these results, we suggest that epithelial migration is significantly altered by differentiation status. Thus, efforts to improve TETG epithelialization should use model systems that faithfully-represent the differentiation state of the native tissue.

GPER mediates differential effects of estrogen on colon cancer cell proliferation and migration under normoxic and hypoxic conditions.

The estrogen receptor ERβ is the predominant ER subtype expressed in normal well-differentiated colonic epithelium. However, ERβ expression is lost under the hypoxic microenvironment as colorectal cancer (CRC) malignancy progresses. This raises questions about the role of signalling through other estrogen receptors such as ERα or G-protein coupled estrogen receptor (GPER, GPR30) by the estrogen 17β-estradiol (E2) under hypoxic conditions after ERβ is lost in CRC progression. We tested the hypothesis that E2 or hypoxia can act via GPER to contribute to the altered phenotype of CRC cells.GPER expression was found to be up-regulated by hypoxia and E2 in a panel of CRC cell lines. The E2-modulated gene, Ataxia telangiectasia mutated (ATM), was repressed in hypoxia via GPER signalling. E2 treatment enhanced hypoxia-induced expression of HIF1-α and VEGFA, but repressed HIF1-α and VEGFA expression under normoxic conditions. The expression and repression of VEGFA by E2 were mediated by a GPER-dependent mechanism. E2 treatment potentiated hypoxia-induced CRC cell migration and proliferation, whereas in normoxia, cell migration and proliferation were suppressed by E2 treatment. The effects of E2 on these cellular responses in normoxia and hypoxia were mediated by GPER. In a cohort of 566 CRC patient tumor samples, GPER expression significantly associated with poor survival in CRC Stages 3-4 females but not in the stage-matched male population.Our findings support a potentially pro-tumorigenic role for E2 in ERβ-negative CRC under hypoxic conditions transduced via GPER and suggest a novel route of therapeutic intervention through GPER antagonism.

An Engineered Cell-Instructive Stroma for the Fabrication of a Novel Full Thickness Human Cervix Equivalent In Vitro.

There is a growing interest for developing organotypic cervical models by using primary cervical cells that are able to reproduce the physiological relevant stromal microenvironment and the distinctive histology of the native cervical epithelium. Here for the first time it is reported the production of an organotypic cervical model featured by a scaffold-free stromal tissue resembling the extracellular matrix (ECM) composition and organization of the native counterpart as well as a completely well-differentiated epithelium. To reach this aim, human cervical microtissue precursors have been produced, characterized, and used as functional building units to fabricate a cell-synthesized cervical stroma equivalent by means of a bottom-up approach. Immunotypization, and molecular and morphological analyses reveal the extent of fundamental epithelial biomarkers and the presence of collagen and noncollagenous molecules, demonstrating that the natural tissue architecture and biological characteristics of cervical tissues are reproduced. The results of this study suggest that the bottom-up technology used to produce these 3D human cervical equivalents provides a fully functional organotypic cervical model that may be used as a valuable tool to investigate the epithelial-stromal interactions as well as for testing new therapeutics in vitro.

Intraductal neoplasms of the bile duct. A new challenge to biliary tract tumor pathology.

Invasive biliary tract carcinomas are usually tubular adenocaricnomas with abundant desmoplastic reactions and frequent ductal and periductal invasion at the time of the diagnosis. Recently, several intraductal neoplasms of the bile duct, particularly at a pre-invasive stage, have been recognized. They include intraductal papillary neoplasm of the bile duct (IPNB), biliary intraepithelial neoplasm (BilIN), and others, such as intraductal tubulopapillary neoplasm (ITPN) of the bile duct. IPNBs are grossly visible predominantly intraductal-growing papillary neoplasms covered by well-differentiated neoplastic epithelium with fine fibrovascular cores in the dilated bile ducts. Regarding their similarities to intraductal papillary mucinous neoplasm of the pancreas (IPMN) of main pancreatic duct type, some IPNBs resemble IPMN ("pancreatic type"), while others are only somewhat similar or variably different from IPMN ("non-pancreatic type"). Some IPNBs develop via a common oncogenic signaling pathway, and others, particularly those of intestinal type, frequently show GNAS mutations, as in IPMN. BilINs are a microscopically recognizable flat or micropapillary pre-invasive neoplasm and are presumed to precede conventional nodular-sclerosing cholangiocarcinomas. ITPN of the bile duct is a rare neoplasm composed of densely packed tubular glands. These three types of neoplasms are not infrequently associated with invasive adenocarcinoma. Pre-invasive intraglandular neoplasms of the peribiliary glands, another epithelial system in the biliary tree, have been also reported. Further characterization of these intraductal and intraglandular neoplasms of the bile duct is needed to overcome devastating invasive biliary tract carcinoma.