Molecular phenotyping reveals the identity of Barrett's esophagus and its malignant transition.

The origin of human metaplastic states and their propensity for cancer is poorly understood. Barrett's esophagus is a common metaplastic condition that increases the risk for esophageal adenocarcinoma, and its cellular origin is enigmatic. To address this, we harvested tissues spanning the gastroesophageal junction from healthy and diseased donors, including isolation of esophageal submucosal glands. A combination of single-cell transcriptomic profiling, in silico lineage tracing from methylation, open chromatin and somatic mutation analyses, and functional studies in organoid models showed that Barrett's esophagus originates from gastric cardia through c-MYC and HNF4A-driven transcriptional programs. Furthermore, our data indicate that esophageal adenocarcinoma likely arises from undifferentiated Barrett's esophagus cell types even in the absence of a pathologically identifiable metaplastic precursor, illuminating early detection strategies.

Pathogenesis and Cells of Origin of Barrett's Esophagus.

In patients with Barrett's esophagus (BE), metaplastic columnar mucosa containing epithelial cells with gastric and intestinal features replaces esophageal squamous mucosa damaged by gastroesophageal reflux disease. This condition is estimated to affect 5.6% of adults in the United States, and is a major risk factor for esophageal adenocarcinoma. Despite the prevalence and importance of BE, its pathogenesis is incompletely understood and there are disagreements over the cells of origin. We review mechanisms of BE pathogenesis, including transdifferentiation and transcommitment, and discuss potential cells of origin, including basal cells of the squamous epithelium, cells of esophageal submucosal glands and their ducts, cells of the proximal stomach, and specialized populations of cells at the esophagogastric junction (residual embryonic cells and transitional basal cells). We discuss the concept of metaplasia as a wound-healing response, and how cardiac mucosa might be the precursor of the intestinal metaplasia of BE. Finally, we discuss shortcomings in current diagnostic criteria for BE that have important clinical implications.

Histological features of gastric cardia in adults: an autopsy study.

BACKGROUND AND AIMS: The existence, histology and origin of gastric cardiac mucosa are controversial. The aim of the present study was to determine the existence, histological characteristics and length of cardiac mucosa and to correlate these features with the patients' age and the presence of inflammation in the gastric cardia and/or esophagus. METHODS; The cardiac mucosa within the whole esophagogastric junction was histologically analyzed in 38 consecutive autopsy specimens and measured in 24 cases. RESULTS: The cardiac mucosa was identified in all specimens from all cases, with a mean length of 6.7 mm, range 0.927-19.5 mm. In the majority of cases, the length of cardiac mucosa was less than 10 mm (87.5%) and greater than 5 mm (71%). Cardiac mucosa was composed of a combination of pure mucous glands and mucous glands with parietal cells in 74% of cases, and only of mucous glands with parietal cells in 26% of cases. Carditis was recorded in 23.7% cases and reflux esophagitis in 15.8%. The length of cardiac mucosa was not significantly different between cases with and without carditis (p>0.05), between those with and without esophagitis (p>0.05), and between age groups older and younger than 60 years (p>0.05). CONCLUSION: In the adult population, a short histological segment of gastric cardia was consistently present as a normal histological structure. The type, length and circumferential presence of cardiac mucosa were not significantly associated with carditis, esophagitis or age.

Esophageal pH exposure and epithelial cell differentiation.

It is proposed that epithelial changes induced by gastroesophageal reflux disease are related to the pH environment of the esophageal lumen. We hypothesized that the various types of esophageal epithelium are associated with specific pH environments that induce their formation. The aim of this study was to compare the luminal pH environment to the histology of the distal esophageal epithelium in patients with gastroesophageal reflux disease. A total of 197 symptomatic patients with increased esophageal acid exposure on 24-hour pH monitoring were grouped according to the histology based on biopsies from the distal esophagus: 17 with squamous epithelium, 126 with cardiac epithelium (CE), and 54 with Barrett's epithelium (BE). All were free of Helicobacter pylori infection and monitored off acid suppression therapy. Acid exposure was expressed as the percent of time the luminal pH was at intervals of 0-1, 1-2, 2-3, 3-4, 4-5, 5-6, and 6-7 over a 24-hour period. Patients with BE spent significantly more time at pH intervals 2-3, 3-4, and 4-5 than those with CE. This pattern switched at pH interval 5-6, where patients with cardiac mucosa spent more time than those with BE. Patients with squamous and CE had similar pH exposure at all intervals. Patients with BE have significantly longer exposure time at the pH interval of 2 to 5 compared to those with cardiac and squamous epithelium. This suggests that the exposure of stem cells to a luminal pH between 2 and 5 may trigger the differentiation of CE into intestinalized CE.

Expression of estrogen receptor-alpha protein in the rat digestive tract.

The current study evaluates the expression of estrogen receptor-alpha (ER-alpha) protein in the digestive tract and other organs using immunohistochemistry in male and female intact rats. As a result, the expression of ER-alpha protein was intensively immunoreactive in the nuclei of squamous epithelium of the forestomach connected to the limiting ridge and the anus connected to the anorectal junction. Rat ER-alpha mRNA signals were also detected in the epithelium of the limiting ridge using in situ hybridization. The incidence of ER-alpha protein in the limiting ridge decreased with age in both males and females. The incidence of ER-alpha protein in the anorectal junction strongly decreased with age in males, although the incidence did not decrease with age in females. In conclusion, it was suggested that estrogen may be involved in the proliferation and differentiation of these cells in the limiting ridge of the stomach and anorectal junction of rats.

Localization of TFF3 peptide in human esophageal submucosal glands and gastric cardia: differentiation of two types of gastric pit cells along the rostro-caudal axis.

TFF3 (trefoil factor family 3), which is a major secretory product of the gastric antrum and the intestine, but which is nearly absent in the gastric corpus, plays a key role in the maintenance of mucosal integrity. Here, we have systematically investigated TFF3 expression in the esophagus and gastric cardia by the use of reverse transcription/polymerase chain reaction (RT-PCR) analysis and immunohistochemistry. Synthesis of TFF3, but not TFF1 or TFF2, is detectable in esophageal submucosal glands. The stratified squamous epithelium is devoid of TFF synthesis. Prominent TFF3 expression starts at the Z-line with a sharply decreasing gradient toward the cardia. Immunohistochemistry has localized TFF3 to surface mucous cells of the proximal cardia. TFF3 distribution differs characteristically from that of TFF1 (secreted primarily by superficial surface mucous cells), whereas TFF3, together with the mucin MUC5AC, is also found in deeper lying cells toward the isthmus. This is the first report of TFF3 as a typical secretory peptide of esophageal submucosal glands and gastric cardia. The different expression patterns of TFF3 and TFF1 in the cardia suggest a stepwise maturation of surface mucous cells from TFF3/MUC5AC-positive cells close to the isthmus to TFF1/TFF3/MUC5AC-positive cells at the pit. The gradient of TFF3 expression along the gastric rostro-caudal axis defines two types of gastric pit cells: those secreting TFF3 in the cardia and the antrum and those nearly devoid of TFF3 synthesis in the corpus. This indicates the special requirement, particularly of the esophagogastric junction, for TFF3-triggered protection and repair.

A comparative analysis by SAGE of gene expression profiles of Barrett's esophagus, normal squamous esophagus, and gastric cardia.

BACKGROUND & AIMS: The metaplastic process in which the normal squamous epithelium of the distal esophagus is replaced by columnar-lined epithelium, known as Barrett's esophagus (BE), is poorly understood. The aim of this study was to define, analyze, and compare transcription profiles of BE, normal cardia epithelium, and squamous epithelium to gain more insight into the process of metaplasia and to identify uniquely expressed genes in these epithelia. METHODS: Serial analysis of gene expression was applied for obtaining transcription libraries of biopsy specimens taken from a BE-affected patient with intestinal type of metaplasia and from normal squamous and gastric cardia epithelia. Validation of results by reverse-transcription polymerase chain reaction and immunoblotting was performed using tissues of 20 patients with BE. RESULTS: More than 120,000 tags were sequenced. Between BE and squamous 776, and between BE and gastric cardia 534 tags were significantly differentially expressed (P < .05, pairwise comparison). In contrast, squamous compared with gastric cardia epithelia showed significant differential expression of 1316 tags. The most up-regulated genes in BE compared with squamous epithelium were trefoil factors, annexin A10, and galectin-4. Each of the epithelia showed a unique cytokeratin expression profile. CONCLUSIONS: This study provides a comparison of the transcriptomes of BE, squamous epithelium, and gastric cardia epithelium. BE proves to be an incompletely differentiated type of epithelium that shows similarities to both normal squamous and gastric cardia epithelia. In addition, several uniquely expressed genes are identified. These results are a major advancement in understanding the process of metaplasia that leads to BE.

On the origin of cardiac mucosa: a histological and immunohistochemical study of cytokeratin expression patterns in the developing esophagogastric junction region and stomach.

AIM: To examine the fetal and neonatal esophagogastric junction region (EGJ) histologically for the presence of an equivalent to adult cardiac mucosa (CM); to study the expression patterns of all cytokeratins (CK) relevant to the EGJ during gestation; to compare the CK profile of the gestational and the adult EGJ; and to determine the degree of development in the adult EGJ histology and CK profile during gestation. METHODS: Forty-eight fetal autopsy specimens of the EGJ were step-sectioned and stained with hematoxylin and eosin (H and E) to select sections showing the mucosal lining. Immunohistochemistry for CK5, 7, 8, 13, 18, 19, and 20 was performed. Antibody staining was then graded for location, intensity, and degree. RESULTS: The distal esophagus was lined by simple columnar epithelium from 12-wk gestational age (GA). The proximal part of this segment consisted of mucus-producing epithelium, devoid of parietal cells. CK5 and 13 were present exclusively in multilayered epithelia and CK8, 18, and 19 predominantly in simple columnar epithelium. There were no differences in the frequencies of the co-ordinate CK7+/20+ and the CK7-/20- immunophenotypes between different locations. The prevalence of the CK7+/20- immunophenotype decreased, and that of the CK7-/20+ immunophenotype increased significantly from the distal esophagus to the distal stomach. CONCLUSION: Fetal columnar-lined lower esophagus (fetal CLE) may be the equivalent and precursor of the short segments of columnar epithelium found in the distal esophagus of some normal adult subjects. Esophageal simple columnar epithelium without parietal cells (ESN) may be the precursor of adult CM. The similarities between the fetal and adult EGJ and stomach CK expression patterns support the conclusion that adult CM has an identifiable precursor in the fetus. This would then indicate that at least a part of the adult CM has a congenital origin.

Characterization of receptors for two Xenopus gastrointestinal tachykinin peptides in their species of origin.

Two tachykinin peptides, bufokinin and Xenopus neurokinin A (X-NKA) were recently isolated from Xenopus laevis. In this study we investigated the tachykinin receptors in the Xenopus gastrointestinal tract. In functional studies using stomach circular muscle strips, all peptides had similar potencies (EC50 values 1-7 nM). The rank order of potency to contract the intestine was physalaemin (EC50 1 nM)> or =bufokinin (EC50 3 nM)>substance P (SP)> or =cod SP>NKA>>X-NKA (EC50 1,900 nM). No maximum response could be obtained for [Sar9,Met(O2)11]SP, eledoisin and kassinin. In stomach strips, the mammalian tachykinin receptor antagonists RP 67580 (NK1) and MEN 10376 (NK2) had agonistic effects but did not antagonize bufokinin or X-NKA. In intestinal strips, RP 67580 (1 microM) reduced the maximal response to X-NKA but not bufokinin, while MEN 10376 was ineffective. [125I]BH-bufokinin bound with high affinity to a single class of sites, of KD 213+/-35 (stomach) and 172+/-9.3 pM (intestine). Specific binding of [125I]BH-bufokinin was displaced by bufokinin> or =SP>NKA> or =eledoisin approximately kassinin>X-NKA, indicating binding to a tachykinin NK1-like receptor. Selective tachykinin receptor antagonists were weak or ineffective. Other iodinated tachykinins ([125I]NKA and [125I]BH-eledoisin) displayed biphasic competition profiles, with the majority of sites preferring bufokinin rather than X-NKA. In conclusion, there is evidence for two different tachykinin receptors in Xenopus gastrointestinal tract. Both receptors may exist in stomach, whereas the bufokinin-preferring NK1-like receptor predominates in longitudinal muscle of the small intestine. Antagonists appear to interact differently with amphibian receptors, compared with mammalian receptors.

Observations on aggregated lymphoid nodules in the cardiac glandular areas of the Bactrian camel (Camelus bactrianus).

Aggregated lymphoid nodules are an important part of the gut-associated lymphoid tissue (GALT). They are mainly distributed in the ileum and appendix of animals and humans but their distribution in the cardiac glandular area has not been reported. A study of stomach histology in the Bactrian camel has revealed that the nodules are distributed as a band-like region along the ventral wall of the stomach neck, at the beginning of the cranial enlargement and on the lesser curvature. The mucous folds are thicker in these regions than where there are no aggregated lymphoid nodules. The nodules appeared similar to ileal aggregated lymphoid nodules found in other animals and consisted of typical polymorphological lymphatic nodules arranged in a single continuous row occupying the submucosa and forming mucosal folds together with the mucous membrane. The whole mucous membrane with cardiac glands, diffuse lymphatic tissue and solitary lymphoid nodules in the lamina propria were found to cover the aggregated lymphoid nodule regions, but some nodules with a typical corona extended into the lamina propria and were covered with follicle-associated epithelium devoid of cardiac glands. These findings indicate that the stomach of the Bactrian camel possesses not only a special structure of digestion but also has characteristic immunological morphology.

Immunohistochemical study of neuroendocrine cells at the gastric cardia mucosa.

BACKGROUND/AIMS: The gastric cardia mucosa is a narrow band of tissue between the oesophagus and the stomach. The physiological role of this tissue is unknown. This study examined the presence and characteristics of neuroendocrine cells at this site. METHODS: Biopsy samples were obtained from across normal appearing squamocolumnar junctions. The cardiac mucosa was defined as the presence of special type mucosa composed of mucous secreting glands in the immediate vicinity of oesophageal squamous epithelium. Biopsy specimens were stained with haematoxylin and eosin, alcian blue (pH 2.5) periodic acid Schiff, and modified Giemsa. The chromogranin A and Fontana-Masson stains were used to identify neuroendocrine cells, which were also stained immunohistochemically for gastrin, serotonin, glucagon, pancreatic polypeptide, somatostatin, and vasoactive intestinal peptide. RESULTS: Chromogranin positive cells were seen in 18 cases with adequate biopsy specimens from the gastric cardia mucosa. These cells were all serotonin positive, but stains for other peptide hormones remained negative. Serotonin positive cells were detected only at the base of foveolae at the periphery of mucous secreting cardiac glands, giving a microscopic appearance resembling that of endocrine cells at the gastric antrum. The presence and numbers of serotonin positive cells did not correlate with chronic inflammation or intestinal metaplasia of the cardiac mucosa. These cells were seen both in Helicobacter pylori positive and negative patients. CONCLUSIONS: Serotonin positive cells appear to be the sole neuroendocrine cell type at the gastric cardia mucosa. These cells may have a role in regulating the physiology of the gastric cardia mucosa and the lower oesophageal sphincter.

Laminins and TGF-beta maintain cell polarity and functionality of human gastric glandular epithelium.

The human gastric glandular epithelium produces a gastric lipase enzyme (HGL) that plays an important role in digestion of dietary triglycerides. To assess the involvement of extracellular matrix components and transforming growth factor-beta1 (TGF-beta1) in the regulation of this enzymic function, normal gastric epithelial cells were cultured on collagen type I, Matrigel, and laminins (LN)-1 and -2 with or without TGF-beta1. Epithelial morphology and HGL expression were evaluated using microscopy techniques, enzymic assays, Western blot, Northern hybridization, and RT-PCR. A correlation was observed between the cell polarity status and the level of HGL expression. TGF-beta1 alone or individual matrix components stimulated cell spreading and caused a downfall of HGL activity and mRNA. By contrast, Matrigel preserved the morphological features of differentiated epithelial cells and maintained HGL expression. The combination of LNs with TGF-beta1 (two constituents of Matrigel) exerted similar beneficial effects on epithelial cell polarity and evoked a 10-fold increase of HGL levels that was blunted by a neutralizing antibody against the alpha(2)-integrin subunit and by mitogen-activated protein kinase (MAPK) inhibitors PD-98059 (p42/p44) or SB-203580 (p38). This investigation demonstrates for the first time that a powerful synergism between a growth factor and basement membrane LNs positively influences cell polarity and functionality of the human gastric glandular epithelium through an activation of the alpha(2)beta(1)-integrin and effectors of two MAPK pathways.

Phenotypic identity of gastric mucous neck cells and mucous cells of cardiac, pyloric, and Brunner's glands.

AIM: To investigate the tissue specificity of a novel monoclonal antibody raised to a tissue fraction of normal human liver and which identified certain cells of gastric and duodenal mucosa. METHODS: A total of 155 samples of various tissues obtained from 100 surgical specimens were fixed in cold ethanol-paraformaldehyde, embedded in paraffin wax, and 3 microns sections were studied by immunohistochemical and lectin staining procedures. RESULTS: Immunohistochemical staining showed a major tissue specific component which was strongly expressed by mucous neck cells of the body of the stomach, glands of the cardia and pyloric antrum, and by Brunner's glands. Staining for antigen in the periductal glands of normal major biliary and pancreatic ducts was variable and relatively weaker. It was not detected elsewhere in normal intestine or in the other normal tissues tested. Barrett's mucosa of gastric cardia type, and pyloric gland metaplasia in the gall bladder and small bowel affected with Crohn's disease stained for the antigen. The tissue distribution of the antigen was identical with that of a glycoprotein, demonstrated by an induced affinity for concanavalin A following treatment of tissue sections with periodic acid. The antigen was not sensitive to sialidase. CONCLUSIONS: The tissue component identified (designated here as antigen D10) seems to be characteristic of certain differentiated epithelial cells derived from that part of foregut giving rise to stomach, duodenum, and biliary and pancreatic ducts. The antibody will be of use in investigating pathological processes involving tissue differentiation at these sites, and in the oesophagus and intestines.

[Quantitative microscopic characteristics of cardial glands in in the human esophagus in postnatal ontogenesis]. / Kolichestvennye mikroskopicheskie kharakteristiki kardial'nykh zhelez pishchevoda cheloveka v postnatal'nom ontogeneze.

Microscopical investigation of oesophagus, obtained from corpses of 33 men and 33 women has been carried out (staining with hematoxylin and eosin, van Gieson). The cardial glands have been revealed in 92.4% of cases in the inferior and in 4.6% of cases in the superior third of the oesophageal wall. They have not been revealed in the superior third of the oesophageal wall. They have not been revealed in its medial third. Amount of acini in the section is essentially changeable. In elderly and old persons the ducts of the glands often form ampullar dilatations. The acinar areas on the section remain stable during the greatest++ period of the postnatal ontogenesis and only during old age they decrease slightly. The proper plate of the mucous membrane in the inferior part of the oesophagus in the zone, where the cardial glands are situated, is always thicker than in the area free from the acini. Close interrelations have been revealed between the cardial glands and lymphoid tissue of the oesophageal wall. The intensity of the glandular-lymphoid interrelations is insignificant in newborns and in children of suckling age. It is maximal in persons of mature and elderly age. Remaining at a sufficiently high level, the glandular-lymphoid associations in old persons are manifested in a less degree than in the previous age groups. No difference in organization of the cardial glands has been revealed in the superior and inferior parts of the oesophageal wall, as well as any sex peculiarities.

Phylogenetic diversity of cellular organization in the cardia of muscoid flies (Diptera: Schizophora).

In each of 30 dipteran species, representing 13 acalyptrate and 7 calyptrate families, the cardia is formed from specialized cells at the junction between foregut and midgut. Foregut epithelium forms the stomodeal valve; midgut epithelium envelops the valve to form the cardia's outer wall. Cytological characteristics within these epithelia differ from region to region and from species to species. Since the cardia secretes the peritrophic membrane, cardias with diverse patterns of cellular differentiation may be expected to produce peritrophic membranes with similarly diverse properties. Close relatives often share more details of cardia structure than do distantly related taxa. Within the monophyletic Calyptratae, a common pattern of cellular differentiation includes three distinct zones of columnar midgut cells enclosing a flanged stomodeal valve. Among species in the paraphyletic Acalyptratae, midgut typically includes a single zone of tall columnar cells, while the valve may be spheroidal, cylindrical, conical, or flanged. The correlation of phylogenetic distance with divergence in cardia organization implies a strong influence of ancestry upon current structure, regardless of current diet. However, at least some of the observed diversity in cardia structure is associated with dietary divergence. Calyptrate flies with derived blood-feeding behavior display cellular differentiation that is simplified from that seen in calyptrate relatives with less specialized feeding habits. This evolutionary modification suggests that cardia organization and hence peritrophic membrane structure can adapt to dietary changes, with possible significance for the spatial organization of digestive processes and interactions with ingested microorganisms.

[Endocrine cells in the cardial glands of the esophagus in man]. / Endokrinnye kletki kardial'nykh zhelez pishchevoda cheloveka.

Distribution of endocrine cells in composition of the secretory epithelium of the cardial glands of the human esophagus in both sex and at various age has been investigated. In spiral paraffin slices the endocrine cells have been revealed by means of different silver impregnation methods (after Grimelius, Masson--Hamperl, Sevier--Munger), Sevke technique, ferry-ferrocyanide method. Some cells have been revealed, which according to the specific signs of their granule staining resemble very much G-, EC-, ECL-cells of the stomach. They can be triangular, flatten or polygonal and are stained in the cardial gland epithelium as single diffuse cells, or as groups of cells. Staining of the slices with aldehyde-fuchsin in various modifications reveals dark cells with dark-violet granules and lighter cells with acidophilic granules. Sometimes among these cells certain cells with light-violet cytoplasm are revealed. All these cells can be arranged both in composition of the secretory epithelium of the glands and in conglomerates of cells, resembling pancreatic islands. According to their tinctorial properties they resemble A-, B-, D-cells of these islands.

Ultrastructure of the peritrophic membrane-secreting cells in the cardia of the blowfly, Lucilia cuprina.

Scanning and transmission electron microscopy are used to reveal the internal anatomy and ultrastructure of the cardia which is the source of the triple layered peritrophic membrane in the blowfly Lucilia cuprina. Within the cardia, rings of secretory cells (formation zones) and non-secretory tissue (valvula cardiaca) interlock to secrete and mould the layers of membrane. Formation zone cells have abundant rough endoplasmic reticulum, Golgi and secretory vesicles. A portion of midgut just posterior to the formation zone is covered by close-packed microvilli connected by septate-like junctions. The cuticle-lined valvula cardiaca is rich in smooth endoplasmic reticulum, glycogen and microtubules. The oesophageal cuticle is unusual in containing tubular structures. The ultrastructural features of the separate components of the cardia are discussed in terms of their secretory and non-secretory roles; modified midgut cells secrete chitin and protein whereas modified foregut tissue (valvula cardiaca) appears to be adapted to provide structural integrity (extensive junctions, microtubules), movement (muscles, possibly microtubules), a store of energy (glycogen deposits) and possibly a lipidic secretion (from smooth endoplasmic reticulum) to lubricate the passage of the membranes.

Peptic cells.

Immunocytochemical studies have demonstrated the existence of five different types of peptic cells in man. These are chief and mucus neck cells in fundic gland mucosa, the pyloric glands in antral mucosa, and the cardiac and Brunner's glands. There are two immunochemically distinct groups of pepsinogens, pepsinogen group I (PG I) and pepsinogen group II (PG II). Chief cells and mucous neck cells in fundic gland mucosa contain both PG I and PG II. Cardiac gland cells, pyloric gland cells and cells in Brunner's glands are clear staining (with hematoxylin and eosin) and usually contain only PG II. On occasion, faint positivity with PGI antiserum may be found in clear staining pyloric gland cells. Gastric gland heterotopia and metaplasia of fundic or pyloric type may be found anywhere in the gastrointestinal tract, but most often in the distal esophagus and duodenal bulb. Heterotopic and metaplastic gastric cells contain pepsinogens similar to the normal peptic cells in the stomach: chief-type cells contain both PG I and PG II, and clear staining cells only PG II. In the presence of hydrochloric acid, the production of pepsinogens may cause local peptic digestion outside of the stomach.