Ultrastructural features of the nasal mucosa after massive removal of all soft tissues in the inferior turbinate hypertrophy

The nasal respiratory mucosa plays a key role in conditioning the inhaled air and in regulating the immune response against foreign particles. Many authors recom- mend mucosal sparing techniques for the surgical management of the inferior turbinate hypertrophy, such as laser surgery, radiofrequency electrocautery, cryosurgery, etc. In spite of their conservative purposes, ultrastructural studies demonstrate that these methods produce irreversible changes in the nasal mucosa. The aim of this study was to analyze ultrastructural features of the healing process after Microde- brider Assisted Turbinoplasty, alternative procedure that provides a radical interven- tion with removal of both mucosal and submucosal soft tissues of the inferior turbi- nate. Nasal mucosa biopsies from 7 patients (mean age 42+10) affected with inferior turbinate hypertrophy were taken before operation and 4 months after surgery and compared with 2 unaffected controls of the same age. Preoperative samples, routinely processed for Transmission Electron Microscopy (1), displayed a number of ultrastructural changes compared to normal controls such as disrupture of tight junctions, disappearance of pseudostratification, loss of cilia, fibrosis, and abundant inflamma- tory infiltrates. By contrast, in postoperative samples the nasal mucosa showed a normal appearance, with restoration of the pseudostratified ciliated pattern, intercellular connections and normal cellular morphology; collagen showed a normal organization in bundle and fibrosis and submucosal oedema were considerably reduced. In the light of these observations and previous Scanning Electron Microscopy findings (2), we can conclude that Microdebrider Assisted Turbinoplasty is a surgical procedure able to radically remove the inflamed tissue typical of hypertophic rynopathy and, consequently, to allow the resident stem cells to re-epithelize the nasal mucosa and restore its normal function

Embryoid body in vitro formation from human amniotic fluid stem cells (AFSCs): an ultrastructural study

Amniotic fluid stem cells (AFSCs) harbour the potential to differentiate into cells of any of the three germ layers and to form embryoid bodies (EBs) without inducing teratoma formation (De Coppi et al., 2007; Antonucci et al., 2012). However, no previous findings have been reported regarding embryoid body in vitro development and ultrastructural organization. Thus, this was the aim of our study. Amniotic fluid samples were obtained from women undergoing amniocentesis for prenatal diagnosis at 16-19 weeks of pregnancy after written informed consent and the local ethical committee approval. Human AFSCs were cultured up to the 8th passage and analysed with RT-PCR for the expression of pluripotency markers. Some cellular pools were cultured in suspension in uncoated Petri dishes (hanging drop method) to obtain EB formation. After 5 days of culture, the appearance of EBs of different size was observed with phase contrast microscopy and monitored up to 10-15 days of culture. In parallel, EB standard embedding in paraffin for light microscopy or in epoxy resin for transmission electron microscopy was performed. RT-PCR analysis revealed the presence of classical markers of pluripotency (OCT4, NANOG, SOX2) in AFSCs at the 2th-8th passage, whereas specific markers of the three embryonic germ layers were detected in EB specimens. Morphological assays of three-dimensional aggregates demonstrated the presence of solid structures only at the beginning of the culture whereas signs of apoptotic cell death accompanied by the secretion of an amorphous substance were soon detected. These features were preliminary to the development, at later culture time intervals, of an inner hollow cavity surrounded by a crown of flat cells displaying a number of electron dense granules and highly resembling trophoblastic cells

A morpho-functional analysis of embryoid body-like structures from human amniotic fluid-derived stem cells (AFSCs) unselected for c-kit

Human AFSCs, a novel class of stem cells sharing characteristics of both embryonic and adult stem cells, harbour high proliferative capacity and high differentiation potential and do not raise the ethical concerns associated with human embryonic stem cells (ESCs). The formation of three-dimensional aggregates known as embryoid bodies (EBs) is the main step in the differentiation of pluripotent embryonic stem cells. The purpose of this study was to investigate whether human AFSCs, unselected for c-kit, have features of pluripotency. With this aim, we evaluated both AFSC ability to form in vitro EB structures and transcriptional profiles of genes typically expressed in human ESCs. Total AFSCs were cultured in suspension in uncoated Petri dishes for EB formation, whose incidence was assessed in 5 independent experiments. EB-like structures were observed and morphometrically analysed under a LEICA phase contrast microscope equipped with a CoolSnap videocamera. A number of samples were processed for alkaline phosphatase (AP) or haematoxylin-eosin staining, immunofluorescence and transmission electron microscopy to follow-up morphology and markers of pluripotency. As to the expression studies, RNA was extracted from AFSCs at the 3th, 4th, 5th and 8th passage in culture and the presence of ESC and primordial germ cell (PGC) specific markers was assessed with RT-PCR. As early as after 5 days of culture we were able to observe the formation of EB-like solid structures of different size progressively increasing at later time intervals of incubation in cell culture medium (10-15 days). At these later time points EB aggregates showed the presence of an internal cavity and were surrounded by a wide cohort of bigger cells detaching from them. Both early and late time EBs were positive for alkaline phosphatase (AP) staining and specific markers of pluripotency (OCT4 and SOX2). The parallel analysis of AFSCs with RT-PCR demonstrated the presence of ESC and PGC specific gene transcripts and, moreover, the expression of alternatively spliced genes also detectable in EB cells. These findings demonstrate that AFSCs are a new and powerful biological system to recapitulate the three-dimensional and tissue level contexts of in vivo development

Engraftment of human amniotic fluid stem cells (AFSCs) in calvarial bone of immunodeficient mice

AFSCs represent an attractive cell model for transplantation therapy due to the lack of significant immunogenicity, tumorigenicity and ethical issues (De Coppi et al., 2007). Although AFSCs have been investigated for bone repair, the cellular distribution and post-implantation viability remain key issues (Dupont et al., 2010). The present study was aimed at investigating whether AFSCs could improve bone healing in a calvarial defect model using immunodeficient mice. For this purpose AFSCs were transfected with a lentiviral vector expressing a ubiquitously directed red fluorescent protein-cherry. For in vivo experiments a critical size (3.5 mm) calvarial defect was developed in NOD scid gamma (NSG) immunodeficient mice. Human AFSCs were expanded in vitro and transfected at the 1st passage, then transplanted in vivo at the lesion sites after being loaded on HEALOS® scaffold (cross-linked collagen fibers fully coated with hydroxyapatite) appropriately shaped to cover the bone lesion. The calvarial defect was filled with the scaffold alone in control mice. Six weeks after implantation all animals were subjected to a skull X-ray before being sacrificed. Calvarial bone specimens were fixed in paraphormaldehyde, cryopreserved with sucrose and embedded in Cryomatrix TM resin. Sections were observed under fluorescence microscopy to detect the cherry-red signal, and then stained with haematoxylin-eosin solution to better analyze histological structures. Radiography scans of ex vivo bone explants demonstrated the presence of qualitatively and quantitatively mineralized tissue levels in the defect. Light microscopy observations revealed a major fibrous reaction in mice specimens treated with the scaffold supplemented with AFSCs compared with mice treated with the cell-free scaffold. The presence of cherry-positive AFSCs was recognized in the newly formed fibrous bone often around the scaffold and close to newly formed vessels. Our findings indicate that undifferentiated AFSCs seeded on a collagen scaffold can engraft in a host bone contributing to new bone and vessel formation. These preliminary observations pave the way to the use of new bioengineered constructs of stem cell–collagen scaffold for correcting large cranial defects in animal models and human subjects

Ultrastructural analysis reveals differences in the secretory activity among four regions of amniotic membrane

Human Amniotic Epithelial Cells (hAEC) from term placenta are a promising source of stem cells for regenerative medicine. In a previous study we observed histological heterogeneity, together with different expression of pluripotency markers and content in lipid granules among four regions of amniotic membrane (AM). To better investigate cell heterogeneity among different cell populations, we performed an ultrastructural study with Transmission Electron Microscopy. Term placentae from healthy women were collected after caesarean section and AM samples were freshly isolated from four regions: R1 (close to the umbilical cord); R2 (intermediate); R3 (peripheral to the placental disc); R4 (reflected amnion). Ultrastructural analysis revealed an epithelium of variable thickness, cellular shape, amount and type of vesicles in the four regions. The epithelium showed columnar hAEC with increased height in R1 and R3 and a multi-layered organization in R3, whereas it was a monolayer in the other regions. The highest amount of granules and vesicles was observed in R3, although R4 showed granules with a different density. Furthermore, in R1, R3 and R4 we noticed several vesicles of 100-150 nm in diameter, probably exosome-like structures, suggesting a consistent secretory activity. All along its length the epithelium was rich in microvilli both on the side facing the amniotic fluid and in lateral contacts (narrow desmosomal junctions) between cells. This in situ investigation shows for the first time differences in secretory activity and granules appearance along the AM as a proof of its heterogeneity. This could be relevant in clinical applications as the choice of the area could improve the effectiveness of AM/hAEC transplantation

Ultrastructural changes of the intestinal mucosa in Non-Celiac Gluten Sensitivity patients could represent an early indicator of cellular stress

Non-Celiac Gluten Sensitivity (NCGS) is a wide-spreading syndrome and an emerging problem in clinical practice linked to the increase of gluten content in some populations diet. It is characterized by intestinal and/or extra intestinal symptoms that improve or disappear after removing gluten from diet in non-celiac or non-wheat allergic patients [1]. In order to perform and support a precise and early diagnosis, as others recently suggested [2], the aim of this work was to analyze in detail ultrastructural features of the duodenal mucosa. Biopsy specimens were obtained from 10 patients who underwent gastrointestinal endoscopy for a diagnostic check-up at the Department of Gastroenterology of the University of Chieti and prepared for electron microscopy. Semithin sections were blindly observed but only biopsies showing well-shaped intestinal villi were selected for the ultrastructural study, observed with a ZEISS EM109 equipped with a Gatan videocamera. We analyzed: 1) brush border, 2) epithelial cell cytoplasm, 3) cellular junctions and 4) the villus connective axis with respect to inflammatory cell number and vascular alterations, evaluating amount and localization of cellular damages. Interestingly, only 3 of these biopsies, obtained from subjects in which clinical history and diagnosis was uncertain, presented fine spot damage in the epithelium from intestinal villi with an apparently normal morphology. Some epithelial cells showed sever distress such as heterochromatic and nuclei not-round shaped, dilated endoplasmic reticulum, increased number of mitochondria and a messy brush border thinned and reduced in width. Numerous damaged cellular junctions and remarkable basal detachment of cell plasma membranes were observed. These findings pave the way to a deepen characterization of intestinal mucosa from NCGS patients to identify, by means of electron microscopy, potential morphological and functional markers of NCGS. This work was supported by MIUR funds granted to R. Di Pietro and L. Centurione in 2014 and 2015

Regulation of Cancer Cell Responsiveness to Ionizing Radiation Treatment by Cyclic AMP Response Element Binding Nuclear Transcription Factor

Cyclic AMP response element binding (CREB) protein is a member of the CREB/activating transcription factor (ATF) family of transcription factors that play an important role in the cell response to different environmental stimuli leading to proliferation, differentiation, apoptosis, and survival. A number of studies highlight the involvement of CREB in the resistance to ionizing radiation (IR) therapy, demonstrating a relationship between IR-induced CREB family members’ activation and cell survival. Consistent with these observations, we have recently demonstrated that CREB and ATF-1 are expressed in leukemia cell lines and that low-dose radiation treatment can trigger CREB activation, leading to survival of erythro-leukemia cells (K562). On the other hand, a number of evidences highlight a proapoptotic role of CREB following IR treatment of cancer cells. Since the development of multiple mechanisms of resistance is one key problem of most malignancies, including those of hematological origin, it is highly desirable to identify biological markers of responsiveness/unresponsiveness useful to follow-up the individual response and to adjust anticancer treatments. Taking into account all these considerations, this mini-review will be focused on the involvement of CREB/ATF family members in response to IR therapy, to deepen our knowledge of this topic, and to pave the way to translation into a therapeutic context

Supplemental Material, supp - Mapping of the Human Placenta: Experimental Evidence of Amniotic Epithelial Cell Heterogeneity

<p>Supplemental Material, supp for Mapping of the Human Placenta: Experimental Evidence of Amniotic Epithelial Cell Heterogeneity by Lucia Centurione, Francesca Passaretta, Maria Antonietta Centurione, Silvia De Munari, Elsa Vertua, Antonietta Silini, Marco Liberati1, Ornella Parolini, and Roberta Di Pietro in Cell Transplantation</p