Prenatal developmental sequences of the esophageal epithelium in the New Zealand white rabbits: Light and electron microscopic analysis.

Several morphogenetic sequences occur during esophageal development and birth defects occur due to defects in foregut morphogenesis. This work aimed to record the cellular events in the morphogenesis of rabbits' esophageal epithelium. On the 16th day of gestation, the esophageal epithelium varied from stratified ciliated columnar to stratified squamous type. The surface epithelium presented mucous cells with mucigen granules of various sizes occupying their supranuclear cytoplasm. Cytoplasmic vacuolation was evident in all layers of the esophageal epithelium at this age. On the 18th gestational day, some light cells could be detected in the middle portion of the epithelium, while others occupied the whole epithelial length. On the 21st day, mucous cells are more frequently observed at the apical esophageal part as well as at the surface epithelium. Numerous elongated dark cells could be distinguished embedded between the basal cells. On the 24th gestational day the number of the mucous cells reached its peak. Reaching the 30th gestational day, several lamellar bodies, a keratinized layer and mitotic divisions could be demonstrated, and the number of both mucous and dark cells was greatly decreased. Collectively, detection of surface mucous and dark cells together with the non-cornified surface in some regions of the rabbit esophageal epithelium at the end of gestation ensure a postnatal development to reach the adult epithelium essential to sustain the passage of the harsh raw food. Future immunohistochemical studies are recommended to investigate the components of secretions in mucous cells and functional studies to highlight the dark cells significance. RESEARCH HIGHLIGHTS: Esophageal epithelium of fetal rabbit was analyzed by light and transmission microscopy. Surface epithelium presented mucous cells with mucigen granules of various sizes. They reached their maximum number on 24th day then decreased. On the 16th day, cytoplasmic vacuolation was evident in all epithelial layers. On the 21st day, numerous elongated dark cells could be distinguished embedded between the basal cells. Before birth, several lamellar bodies, a keratinized layer and mitotic divisions could be demonstrated, and the number of both mucous and dark cells was greatly decreased.

Pre-gastric secretory epithelium: A light, scanning and transmission electron microscopic study of an epithelial modification of the esophagus in embryonic quails.

The current study investigated epithelial modification of embryonic quail esophagus using gross examination, light microscopy, transmission and scanning electron microscopy. By semithin sections, the pre-gastric modified region had unfolded mucosa, formed epithelial flabs and pockets, and had reduced muscularis mucosae, thin muscular layer, less glandular tissue, and outer esophageal groove. Conversely, the normal esophageal mucosa was folded, had abundant glandular tissue and prominent muscularis mucosae, with two muscular layers; the outer and the inner. The modified epithelium resembled stratified squamous type that had a high affinity for PAS, methylene blue, and PAP stains. Ultra-structural features of the modified esophageal epithelium resembled stratified squamous epithelium and contained hypertrophic Keratinocytes; dark and light. Hypertrophic keratinocytes had RER organized, few ribosomes, and developed loose bundle of cytokeratin compared with squamous keratinocytes. Hypertrophic Keratinocytes synthesize two types of granules; peripherally located small electron-dense granules and large electron-lucent granules. Hypertrophic keratinocytes had peroxisomes that were identified by the crystalline core of the urate oxidase. In conclusion, epithelia modification may have secretory function. Further studies should be carried out to explain the exact function of this type of modified epithelium.

Histological characterization of the gastrointestinal tract of the adult horseshoe crab (Limulus polyphemus) with special reference to the stomach.

The American horseshoe crab (Limulus polyphemus) is one of four extant species in the Order Xiphosura, subphylum Chelicerata, and are evolutionarily more closely related to scorpions and spiders, than crabs. The basic structure, function, and physiology of these invertebrates and their internal organs are not well documented in the literature. In this study, the gastrointestinal system, with a focus on the stomach, of adult L. polyphemus were assessed by gross and histologic methods to further characterize the pyloric valve, the lining of the ventricular lumen, and the muscular tunics of the stomach. Determination of normal anatomical structure of this organ system, along with characterization of the esophagus and intestinal tract, will set a standard against which tissue abnormalities, such as those seen with disease or pathology were to arise, would allow for better interpretation.

Functional morphology of the glandular esophageal pouches of chitons (Mollusca, Polyplacophora).

The esophageal pouches of Chaetopleura angulata and Acanthochitona fascicularis were investigated using light and transmission electron microscopy. These pouches linked to the posterior region of the esophagus are known as sugar glands as they contain a fluid rich in polysaccharide digesting enzymes. They are the second largest glands in the digestive system of chitons, just after the digestive gland. In both species, the pouches contain a dense array of finger-shaped villi. The villi epithelium includes absorptive cells, basophilic secretory cells, mucus-secreting cells, and basal cells. Some absorptive cells were bordered by a dense cover of long microvilli, whereas other absorptive cells had short and sparse microvilli. Absorptive cells contain several lysosomes, mitochondria, peroxisomes, a few small Golgi stacks, some lipid droplets, and large amounts of glycogen. The basophilic secretory cells are characterized by the presence of many electron-dense vesicles, with a glycoprotein content, a large number of rough endoplasmic reticulum cisternae, and a highly developed Golgi apparatus. Mucus-secreting cells are characterized by large vesicles containing acid polysaccharides and wide Golgi stacks. Basal cells that were found at the base of the epithelium in contact with the basal lamina exhibit histological and ultrastructural features of enteroendocrine cells. We suggest that these glandular pouches are involved in extracellular and intracellular digestion, and accumulate lipid and glycogen reserves.

Subcellular Localization of the TFF Peptides xP1 and xP4 in the Xenopus laevis Gastric/Esophageal Mucosa: Different Secretion Modes Reflecting Diverse Protective Functions.

The TFF peptides xP1 and xP4 from Xenopus laevis are orthologs of TFF1 and TFF2, respectively. xP1 is secreted as a monomer from gastric surface mucous cells and is generally not associated with mucins, whereas xP4 is a typical secretory peptide from esophageal goblet cells, and gastric mucous neck and antral gland cells tightly associated as a lectin with the ortholog of mucin MUC6. Both TFF peptides have diverse protective functions, xP1 as a scavenger for reactive oxygen species preventing oxidative damage and xP4 as a constituent of the water-insoluble adherent inner mucus barrier. Here, we present localization studies using immunofluorescence and immunoelectron microscopy. xP1 is concentrated in dense cores of secretory granules of surface mucous cells, whereas xP4 mixes with MUC6 in esophageal goblet cells. Of note, we observe two different types of goblet cells, which differ in their xP4 synthesis, and this is even visible morphologically at the electron microscopic level. xP4-negative granules are recognized by their halo, which is probably the result of shrinkage during the processing of samples for electron microscopy. Probably, the tight lectin binding of xP4 and MUC6 creates a crosslinked mucous network forming a stabile granule matrix, which prevents shrinkage.

Analysis of the green turtle esophagus Chelonia mydas (Linnaeus, 1758), Testudines, Cheloniidae / Análisis del esófago de la tortuga verde Chelonia mydas (Linnaeus, 1758), Testudines, Cheloniidae

The esophagus is a muscular tube whose function is to transport food from the mouth to the stomach and basically contain the same layers as the rest of the digestive tract. The morphological knowledge of structures involved in basic physiological processes in organisms is extremely important in the acquisition of ecological and evolutionary knowledge about species. This study produces morphological information proving the structures found in the green turtle's esophagus. The animals were dissected according to the methodology used by Wyneken, allowing analysis of external and internal morphological characteristics of the esophagus. The samples were processed for microscopic analysis by light and scanning electron microscopy. It was observed that the esophagus presents thin and conical dermal papillae facing the stomach throughout the mucosa to the gastroesophageal portion, decreasing its size at the end of its extension, covered by a thick keratin layer on keratinized pavement epithelial tissue. It is concluded that the esophagus of Chelonia mydas is covered by a layer of conical keratinized dermal papillae important to exert the mechanical and protective function of the mucosa of this organ, considering that the feeding of these animals is extremely abrasive, besides serving as protection so that the food does not return.

El esófago es un tubo muscular cuya función es transportar los alimentos desde la cavidad oral hasta el estómago y, básicamente, contienen las mismas capas que el resto del tracto digestivo. El conocimiento morfológico de las estructuras involucradas en los procesos fisiológicos básicos en los organismos es extremadamente importante en la adquisición de conocimientos ecológicos y evolutivos sobre las especies. Este estudio produce información morfológica que demuestra las estructuras encontradas en el esófago de la tortuga verde. Los animales se disecaron de acuerdo con la metodología utilizada por Wyneken, lo que permitió el análisis de las características morfológicas internas y externas del esófago. Las muestras se procesaron para análisis microscópico mediante microscopía electrónica de barrido y de luz. Se observó que el esófago presenta papilas dérmicas delgadas y cónicas que se enfrentan al estómago a través de la mucosa hasta la porción gastroesofágica, disminuyendo su tamaño al final de su extensión, cubierta por una capa gruesa de queratina sobre el tejido epitelial queratinizado. Se concluye que el esófago de Chelonia mydas está cubierto por una capa de papilas dérmicas queratinizadas cónicas importantes para ejercer la función mecánica y protectora de la mucosa de este órgano, considerando que la alimentación de estos animales es extremadamente abrasiva, además de servir de protección para que la comida no sea devuelta.

Temporospatial localization of telocytes during esophageal morphogenesis in rabbit.

Telocytes (TCs) are CD34 and Vimentin positive (+) immunoreactive stromal cells with a small-sized body and several extremely long telopodes. TCs have been described to provide a mechanical support throughout the tissue by making cellular connections (homo- or hetero) to form a 3D network. Such network can transmit the intercellular signaling. Recently, TCs have been described in the esophageal wall. However, information concerning the role of these cells in esophageal organization and development is rare. Thus, we aimed to record the temporo-spatial localization pattern of TCs during esophageal morphogenesis in rabbit. Embryos and fetuses of New Zealand White rabbits (10th-30th gestational days) were collected. Using CD34 immunostaining, TCs have not been demonstrated in the wall of the developing esophagus till the end of the second third of pregnancy. On 24th gestational day, CD34+ TCs were organized in the adventitia of the esophageal wall specifically in close association with the endothelial cells lining the micro vessels. Later on 26th gestational day, CD34+TCs were additionally expressed in the sub-mucosa and in lamina propria (sub-epithelial). On 28th gestational day, additional CD34+TCs were detected among the smooth muscle bundles of the muscular layer. Reaching the last gestational day, CD34+TCs formed several sheaths in the esophageal wall namely sub epithelial sheath, sub-mucosal, muscular (circular and longitudinal) and inter-muscular sheaths and an outer adventitial one. On the other hand, vimentin immunohistochemistry revealed wider spread TCs positivity in all developmental ages. Presumptively, arrangement of CD34 and vimentin positive TCs in all layers of the developing esophageal wall hypothesizes that TC may play a potential role as a progenitor cell initially in differentiation of the epithelial and muscular precursors and finally in shaping of the various layers of the rabbit esophageal wall during its morphogenesis. TCs are also proposed to be involved in the angiogenesis of the esophageal blood capillaries.

Clinical Translation of Tethered Confocal Microscopy Capsule for Unsedated Diagnosis of Eosinophilic Esophagitis.

Esophagogastroduodenoscopy (EGD) is a widely used procedure, posing significant financial burden on both healthcare systems and patients. Moreover, EGD is time consuming, sometimes difficult to tolerate, and suffers from an imperfect diagnostic yield as the limited number of collected biopsies does not represent the whole organ. In this paper, we report on technological and clinical feasibility of a swallowable tethered endomicroscopy capsule, which is administered without sedation, to image large regions of esophageal and gastric mucosa at the cellular level. To demonstrate imaging capabilities, we conducted a human pilot study (n = 17) on Eosinophilic Esophagitis (EoE) patients and healthy volunteers from which representative cases are presented and discussed. Results indicate that, compared to endoscopic biopsy, unsedated tethered capsule endomicroscopy obtains orders of magnitude more cellular information while successfully resolving characteristic tissue microscopic features such as stratified squamous epithelium, lamina propria papillae, intraepithelial eosinophils, and gastric cardia and body/fundic mucosa epithelia. Based on the major import of whole organ, cellular-level microscopy to obviate sampling error and the clear cost and convenience advantages of unsedated procedure, we believe that this tool has the potential to become a simpler and more effective device for diagnosing and monitoring the therapeutic response of EoE and other esophageal diseases.

Histomorphologic changes of esophageal mucosa in experimental third degree stricture.

INTRODUCTION: Nowadays the level of early and late complications after the operations for esophageal corrosive strictures such as esophago-organ anastomotic leak, development of infections, pneumonia, pleural empyema, mediastinitis, peritonitis, postoperative corrosive stricture development etc. remains rather high. Besides, postoperative mortality rate is high as well - 3.5-30 %. For that reason, an experimental model of esophageal stricture was suggested and ultrastructural mucosal changes in the stricture itself were studied to elaborate the unified pathogenic approach in treatment of esophageal stricture and improvement of its results. The aim of our work was to study the dynamics of ultrastructural changes both in normal esophageal walls and in third degree esophageal stricture Materials and Methods: The experiment was carried out on white male rats weighting 250-300 grams, to whom the third degree esophageal stricture model was created. After layer-by-layer incision of anterior abdominal wall abdominal portion of the esophagus was completely ligated (10 rats). In the control group (6 rats) anterior abdominal wall was opened with its subsequent layered closure. The animals were withdrawn from the experiment on the third day by ketamine overdose, and the samples were taken for ultrastructural study. RESULTS: Electron microscopic study of submicroscopic organization of basal, prickle, superficial epithelial cells in stratified non-squamous epithelium, smooth myocytes of muscle plate and contractile elements in esophageal muscular layer was carried out. Nuclear membrane, membranes of mitochondria, endoplasmic reticulum and cytoplasmic Golgi complex were found to be subjected to focal lysis. The third degree esophageal stricture caused destructive lesions in ultrastructural architectonics of stratified non-squamous epithelium cells, smooth myocytes of muscle plate and contractile elements in esophageal muscular layer of rats. CONCLUSION: Thus, catabolic processes leading to organelle disintegration develop in esophageal cells of rats with third degree stricture.

Ultrastructure of the coelom in the brachiopod Lingula anatina.

The organization of the coelomic system and the ultrastructure of the coelomic lining are used in phylogenetic analysis to establish the relationships between major taxa. Investigation of the anatomy and ultrastructure of the coelomic system in brachiopods, which are poorly studied, can provide answers to fundamental questions about the evolution of the coelom in coelomic bilaterians. In the current study, the organization of the coelom of the lophophore in the brachiopod Lingula anatina was investigated using semithin sectioning, 3D reconstruction, and transmission electron microscopy. The lophophore of L. anatina contains two main compartments: the preoral coelom and the lophophoral coelom. The lining of the preoral coelom consists of ciliated cells. The lophophoral coelom is subdivided into paired coelomic sacs: the large and small sinuses (= canals). The lining of the lophophoral coelom varies in structure and includes monociliate myoepithelium, alternating epithelial and myoepithelial cells, specialized peritoneum and muscle cells, and podocyte-like cells. Connections between cells of the coelomic lining are provided by adherens junctions, tight-like junctions, septate junctions, adhesive junctions, and direct cytoplasmic bridges. The structure of the coelomic lining varies greatly in both of the main stems of the Bilateria, that is, in the Protostomia and Deuterostomia. Because of this great variety, the structure of the coelomic lining cannot by itself be used in phylogenetic analysis. At the same time, the ciliated myoepithelium can be considered as the ancestral type of coelomic lining. The many different kinds of junctions between cells of the coelomic lining may help coordinate the functioning of epithelial cells and muscle cells.

Anterior regeneration after fission in the holothurian Cladolabes schmeltzii (Dendrochirotida: Holothuroidea).

The regeneration of the anterior portion of the body after fission was studied in the holothurian Cladolabes schmeltzii using electron microscopy methods. Following fission, the posterior portion of the digestive tube, cloaca, and respiratory trees remain in the posterior fragment of the body. The regeneration comprises five stages. In the first stage, connective-tissue thickening (an anlage of the aquapharyngeal bulb) occurs on the anterior end between the torn-off ends of the ambulacra. Most of the lost anterior organs developed in the second and third stages. The structures of water-vascular system and nerve ring form through dedifferentiation, proliferation, and migration of cells of the radial water-vascular canals and the radial nerve cords, correspondently. The lost digestive system portion is restored through the formation and merging of two anlagen. The digestive epithelium of the esophagus and pharynx develops from lining cells of microcavities near the central portion of the connective-tissue thickening, which probably migrate from the epidermis. The second gut anlage develops through transformation of the anterior gut remnant portion. The enterocytes partly dedifferentiate, but the epithelium retains integrity. The gut anlage grows down the mesentery and joins the regenerating aquapharyngeal bulb. In the fourth and fifth stages, all lost organs are formed and have nearly normal structure. The regeneration was concluded to occur through morphallactic rearrangements of the remaining parts of organs. Epithelial morphogenesis is the key development mechanism of the digestive, water-vascular, and nervous systems.

Lymphocytic Oesophagitis Preliminary Ultrastructural Observations.

BACKGROUND/AIM: Lymphocytic oesophagitis (LyE) is a newly described entity characterized by a high number of intraepithelial lymphocytes/ high power field (≥40 CD3+IELs/HPF) in the oesophageal epithelium. The aim of the study was to investigate possible ultrastructural changes taking place in LyE at the transmission electron microscopic (TEM) level. MATERIALS AND METHODS: Oesopageal biopsies from seven patients were investigated: four were consecutive patients with LyE, one with reflux oesopagitis, one with eosinophilic oesopagitis (EoE) and one with histologically normal squamous epithelium. RESULTS: In LyE, marked intercellular oedema (spongiosis) and a gamut of regressive changes were found in squamous cells, ranging from cytoplasmic oedema and vacuolization, to total cell disintegration. IELs also showed regressive changes ranging from ballooned, oedematous cytoplasm to signs of intracytoplasmatic disintegration. CONCLUSION: Besides hampered cell nutrition conveyed by spongiosis, putative noxious molecules contained in the intercellular spongiotic oedema might account for the dramatic TEM alterations found in LyE. The present findings provide, for the first time, "inside information" on the ultrastructural alterations taking place in LyE, both in squamous cells and in IELs.

Effect of palonosetron (5HT-3 antagonist) and pantoprazole (proton pump inhibitor) against surgical esophagitis induced by forestomach and pylorus ligation in albino rats.

This study was embarked upon to evaluate the effects of pantoprazole and palonosetron on experimental esophagitis in albino wistar rats. Groups of rats, fasted for 36 h, were subjected to pylorus and forestomach ligation, supervened by treatment with normal saline (3 ml/kg, po, sham control), esophagitis control (3 ml/kg, po), pantoprazole (30 mg/kg, po), palonosetron (0.5 mg/kg, po), and their combination. Animals were sacrificed after 12 h and appraised for the volume of gastric juices, total acidity, free acidity, and esophagitis index. Esophageal tissues were further figured out biochemically for markers of oxidative stress and inflammatory mediators. The combination therapy comparably inhibited the esophagitis index (52.86%), gastric volume (66.04%), free acidity (43.76%), and total acidity (42.60%) in comparison with toxic control. The combination therapy also subsidized the biochemical and inflammatory markers to the purview less than toxic control. The morphological changes were scrutinized by scanning electron microscopy and were observed to demonstrate momentous protection by the amalgamation therapy. Combination therapy with pantoprazole and palonosetron flaunted sententious protection against experimental esophagitis.

Qualitative and Quantitative Analysis of ROS-Mediated Oridonin-Induced Oesophageal Cancer KYSE-150 Cell Apoptosis by Atomic Force Microscopy.

High levels of intracellular reactive oxygen species (ROS) in cells is recognized as one of the major causes of cancer cell apoptosis and has been developed into a promising therapeutic strategy for cancer therapy. However, whether apoptosis associated biophysical properties of cancer cells are related to intracellular ROS functions is still unclear. Here, for the first time, we determined the changes of biophysical properties associated with the ROS-mediated oesophageal cancer KYSE-150 cell apoptosis using high resolution atomic force microscopy (AFM). Oridonin was proved to induce ROS-mediated KYSE-150 cell apoptosis in a dose dependent manner, which could be reversed by N-acetylcysteine (NAC) pretreatment. Based on AFM imaging, the morphological damage and ultrastructural changes of KYSE-150 cells were found to be closely associated with ROS-mediated oridonin-induced KYSE-150 cell apoptosis. The changes of cell stiffness determined by AFM force measurement also demonstrated ROS-dependent changes in oridonin induced KYSE-150 cell apoptosis. Our findings not only provided new insights into the anticancer effects of oridonin, but also highlighted the use of AFM as a qualitative and quantitative nanotool to detect ROS-mediated cancer cell apoptosis based on cell biophysical properties, providing novel information of the roles of ROS in cancer cell apoptosis at nanoscale.

Absolute CT perfusion parameter values after the neoadjuvant chemoradiotherapy of the squamous cell esophageal carcinoma correlate with the histopathologic tumor regression grade.

PURPOSE: To analyze value of the computed tomography (CT) perfusion imaging in response evaluation of the esophageal carcinoma to neoadjuvant chemoradiotherapy (nCRT) using the histopathology as reference standard. METHODS: Forty patients with the squamous cell esophageal carcinoma were re-evaluated after the nCRT by CT examination, which included low-dose CT perfusion study that was analyzed using the deconvolution-based CT perfusion software (Perfusion 3.0, GE). Histopathologic assessment of tumor regression grade (TRG) according to Mandard's criteria served as reference standard of response evaluation. Statistical analysis was performed using Spearman's rank correlation coefficient (r(S)) and Kruskal-Wallis's test. RESULTS: The perfusion CT parameter values, measured after the nCRT in the segment of the esophagus that had been affected by neoplasm prior to therapy, significantly correlated with the TRG: blood flow (BF) (r(S)=0.851; p<0.001), blood volume (BV) (r(S)=0.732; p<0.001) and mean transit time (MTT) (r(S)=-0.386; p=0.014). Median values of BF and BV significantly differed among TRG 1-4 groups (p<0.001), while maximal esophageal wall thickness did not (p=0.102). Median BF and BV were gradually rose and MTT decreased as TRG increased, from 21.4 ml/min/100 g (BF), 1.6 ml/100 g (BV) and 8.6 s (MTT) in TRG 1 group, to 37.3 ml/min/100 g, 3.5 ml/100 g and 7.5 s in TRG 2 group, 81.4 ml/min/100 g, 4.1 ml/100 g and 3.8 s in TRG 3 group, and 121.1 ml/min/100 g, 4.9 ml/100 g and 3.7 s in TRG 4 group. In all 15 patients who achieved complete histopathologic regression (TRG 1), BF was <30.0 ml/min/100 g. CONCLUSIONS: CT perfusion could improve the accuracy in response evaluation of the esophageal carcinoma to nCRT.

Heterogeneous vesicles in mucous epithelial cells of posterior esophagus of Chinese giant salamander (Andrias davidianus).

The Chinese giant salamander belongs to an old lineage of salamanders and endangered species. Many studies of breeding and disease regarding this amphibian had been implemented. However, the studies on the ultrastructure of this amphibian are rare. In this work, we provide a histological and ultrastructural investigation on posterior esophagus of Chinese giant salamander. The sections of amphibian esophagus were stained by hematoxylin & eosin (H&E). Moreover, the esophageal epithelium was observed by transmission electron microscopy (TEM). The results showed that esophageal epithelium was a single layer epithelium, which consisted of mucous cells and columnar cells. The esophageal glands were present in submucosa. The columnar cells were ciliated. According to the diverging ultrastructure of mucous vesicles, three types of mucous cells could be identified in the esophageal mucosa: i) electron-lucent vesicles mucous cell (ELV-MC); ii) electron-dense vesicles mucous cell (EDV-MC); and iii) mixed vesicles mucous cell (MV-MC).

[Age-related peculiarities of mast cell distribution in human esophagus wall].

Mast cells (MCs) were studied quantitatively and qualitatively in the wall of the esophagus (upper, middle and lower portions) of the individuals in the I period of mature age (22-35 years, n = 6) as well as in old and senile persons (61-82 years, n = 10). In all the individuals, the total number of MCs was found to increase from the upper portion of the esophagus towards the lower one. Within the esophageal wall, the total number of MCs decreased in the direction from tunica mucosa towards adventitia. During I period of mature age, the resting MCs filled with granules predominated, while the degranulating MCs were located in the lower esophageal segment. In the individuals of old and senile age, the degranulating MCs were more frequently observed in the upper and lower esophageal segments.

Aberrant DNA hypermethylation reduces the expression of the desmosome-related molecule periplakin in esophageal squamous cell carcinoma.

Periplakin (PPL), a member of the plakin family of proteins that localizes to desmosomes and intermediate filaments, is downregulated in human esophageal squamous cell carcinoma (ESCC). Little is known, however, about the molecular mechanism underlying the regulation of PPL expression and the contribution of PPL loss to the malignant property of the cancer is unclear. We demonstrated that PPL mRNA expression was significantly reduced in ESCC tissues compared with that in normal tissues. Therefore, we hypothesized that CpG hypermethylation is the cause of the downregulation of PPL. Bisulfite-pyrosequencing of 17 cases demonstrated that the frequency of PPL methylation was higher in ESCC tissues than in normal tissues. When human ESCC cell lines were treated with 5-aza-2'-deoxycytidine (5-aza-dC), a DNA-methyltransferase inhibitor, PPL transcription was induced. Human KYSE270 ESCC cells do not stratify under ordinary culture conditions and rarely produce desmosomes; however, the forced expression of PPL promoted cell stratification. PPL induction also promoted adhesion to extracellular matrix but delayed cell migration. The abundance of desmosome-like structures was greatly increased in PPL transfectant as determined by transmission electron microscopy. Very low expression of another desmosome protein EVPL in ESCC, even in PPL transfectant, also supported the significant role of PPL in desmosome formation and cell stratification. Our results first indicate that the downregulation of PPL mediated by DNA hypermethylation, which may play an important role in the loss of ESCC stratification and likely in metastatic phenotype.

Endocytoscopic observation of various esophageal lesions at ×600: can nuclear abnormality be recognized?

Endocytoscopy (ECS) is a novel endoscopic technique that allows detailed diagnostic examination of the gastrointestinal tract at the cellular level. We previously reported that use of ECS at ×380 magnification (GIF-Y0002) allowed a pathologist to diagnose esophageal squamous cell carcinoma (ESCC) with high sensitivity (94.9%) but considerably low specificity (46.7%) because this low magnification did not reveal information about nuclear abnormality. In the present study, we used the same magnifying endoscope to observe various esophageal lesions, but employed digital 1.6-fold magnification to achieve an effective magnification of ×600, and evaluated whether this improved the diagnostic accuracy in distinguishing neoplastic from non-neoplastic lesions.We examined the morphology of surface cells using vital staining with toluidine blue and compared the histological features of 40 cases, including 19 case of ESCC and 21 non-neoplastic esophageal lesions (18 cases of esophagitis, 1 case of glycogenic acanthosis, 1 case of leiomyoma, and 1 case of normal squamous epithelium). One endoscopist classified the lesions using the type classification, and we consulted one pathologist for judgment of the ECS images as 'neoplastic', 'borderline', or 'non-neoplastic'. At ×600 magnification, the pathologist confirmed that nuclear abnormality became evident, in addition to the information about nuclear density provided by observation at ×380. The overall sensitivity and specificity with which the endoscopist was able to predict neoplastic lesions using the type classification was 100% (19/19) and 90.5% (19/21), respectively, in comparison with values of 94.7% (18/19 cases) and 76.2% (16/21), respectively, for the pathologist using a magnification of ×600. The pathologist diagnosed two non-neoplastic lesions and one case of ESCC showing an apparent increase of nuclear density with weak nuclear abnormality as 'borderline'. Among the 21 non-cancerous lesions, two cases of esophagitis that were misdiagnosed by the endoscopist were also misinterpreted as 'neoplastic' by the pathologist. We have shown, by consultation with a pathologist, that an ECS magnification of ×600 (on a 19-inch monitor) is adequate for recognition of nuclear abnormality. We consider that it is feasible to diagnose esophageal neoplasms on the basis of ECS images, and that biopsy histology can be omitted if a combination of increased nuclear density and nuclear abnormality is observed.