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Labeling of apoptotic cells in the gastric mucosa of control (A) and retinoic acid-treated (B) mice. Sections were processed for the TUNEL assay as mentioned in the Material and Methods section and then counterstained with periodic acid Schiff. (A, B): Nuclei of apoptotic cells appear brownish (arrows) and are found at the luminal surface (mucus-secreting pit cells). Note that apoptotic cells are more numerous in (B) compared with (A). Bar = 50 μm.
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The gastric epithelial progenitors proliferate and undergo bipolar migration associated with their differentiation into pit, parietal, and zymogenic cell lineages. Retinoids have long been known to modulate proliferation and differentiation of various renewing epithelia, and the expression of their receptors has been demonstrated in the gastric muc...
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... TUNEL assay was used to detect apoptotic cells in the gastric mucosae of control and retinoic acid-treated mice. As previously reported, pit cells normally undergo degeneration at the luminal surface of the gastric mucosa [17]. However, more pit cells at the luminal surface of reti- noic acid-treated mice underwent apoptosis compared with control mice (Fig. 5). Counts conducted in oxyntic mucosal sections (n = four per mouse) revealed that the number of apoptotic cells was approximately one cell per five glands, whereas in retinoic acid-treated mice, it was approximately three cells per five ...
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Citations
... In the stomachs of mice and humans, the retinoid receptors have been identified in the gastric epithelium and intensified in the area of stem/progenitor cells. It has been also shown that excess retinoic acid stimulates the dynamics of cellular production and differentiation in the mouse gastric glands [19,20]. In humans, while vitamin A (retinol or retinoic acid) is found to inhibit the proliferation of several gastric cancer cell lines Abnormalities in gastric glands occur during the pathogenesis of some gastric disorders, such as gastritis, ulcers, and tumorigenic transformations. ...
... In the stomachs of mice and humans, the retinoid receptors have been identified in the gastric epithelium and intensified in the area of stem/progenitor cells. It has been also shown that excess retinoic acid stimulates the dynamics of cellular production and differentiation in the mouse gastric glands [19,20]. In humans, while vitamin A (retinol or retinoic acid) is found to inhibit the proliferation of several gastric cancer cell lines via antioxidant properties, a meta-analysis based on several case reports and cohort studies have confirmed the inverse relationship between dietary intake of vitamin A and gastric cancer development [21]. ...
... Loss of integrity was also associated with a decrease in epithelial cell proliferation. These findings correlate with our previous study when excess retinoic acid in mice was found to enhance the proliferation of the gastric epithelial stem/progenitor cells [20]. Therefore, the finding that gastric epithelial cell proliferation is inhibited in VAD mice is not unexpected. ...
Junctional epithelia are common sites for pathological transformations. In mice, the stratified epithelium of the forestomach joins the simple glandular epithelium of the cardia at the limiting ridge. We previously demonstrated the expression of vitamin A receptors in the gastric stem/progenitor cells and their progeny and found that excess retinoic acid enhances cellular dynamics of gastric epithelium. This study examines how deficiency of vitamin A would alter gastric epithelial stem cell lineages. Three-week-old mice of both genders were weaned and fed with a vitamin A deficient (VAD) diet for 4 or 8 months. Sex- and weight-matched littermate mice received a standard (control) diet. To label S-phase cells, all mice received a single intraperitoneal injection of 5-bromo-2-deoxyuridine before being euthanized. Stomach tissues were processed for microscopic examination and protein analysis to investigate stem cell lineages using different stains, lectins, or antibodies. The Student’s t-test was used to compare quantified data showing differences between control and VAD groups. Eight-month-vitamin-A deficiency caused enlarged forestomach and overgrowth of the squamocolumnar junction with metaplastic and dysplastic cardiac glands, formation of intramucosal cysts, loss of surface mucosal integrity, increased amount of luminal surface mucus, and upregulation of trefoil factor 1 and H+,K+-ATPase. These changes were associated with decreased cell proliferation and upregulation of p63. In conclusion, vitamin A is necessary for maintaining gastric epithelial integrity and its deficiency predisposes the mouse stomach to precancerous lesions.
... Following incubation with blocking solution, cryosections were incubated for 60 min with different fluorophore-conjugated lectins: Ulex europaeus agglutinin (UEA) I (specific for surface mucous cells), Griffonia simplicifolia (GS) II (for mucous neck cells), or Dolichos biflorus agglutinin (DBA, for parietal cells) [46,47]. Cryosections of cell-containing scaffolds were also incubated overnight with several antibodies specific for H,K-ATPase alpha and beta subunits (for parietal cells), TFF1 (for surface mucous cells), TFF2 (for mucous neck), chromogranin-A (for enteroendocrine cells), and ghrelin (for a subtype of enteroendocrine cells). ...
... Finally, Alexa Fluor (555 or 488)-conjugated avidin was added to visualize antigen-antibody binding sites, using fluorescence Olympus or Nikon Eclipse 80i confocal microscopes (Tokyo, Japan). Cryosections of the cells were also incubated for 60 min with fluorophore-conjugated Ulex europaeus agglutinin (UEA) I lectin (specific for mucous pit cells), Dolichos biflorus agglutinin (DBA) lectin (for parietal cells) or Griffonia simplicifolia (GS)II lectin (for mucous neck cells) (27,28). All lectins were purchased from Sigma (St. Louis, MO, USA). ...
... Such assay will require careful standardization and may even complicate the study, as it is well documented that positive RT-PCR will not always correlate with levels of protein (36) and we have already confirmed mGS cell differentiation into glandular mucous cells using different techniques. Results of both immunohistochemisty and lectin histochemistry demonstrated binding of two very well-characterized biomarkers, anti-TFF2 antibody (29) and GSII lectin (27,28). It is also known that gastric stem cell differentiation into a glandular mucous cells involves increase in cell size due to development of the machinery necessary for production of secretory granules (36). ...
The present study focuses on applying the fracture mechanics approach to the fracture assessment of a cracked member/component strengthened with fiber-reinforced polymer composite stiffeners. The parameters of linear elastic fracture mechanics (LEFM) — the stress intensity factor and the crack opening displacement — are estimated using a finite-element analysis. A metallic plate with an edge crack repaired with fiber-reinforced polymer composite stiffeners is considered in the study. The effects of crack length, debonding length, and adhesive stiffness on the LEFM parameters are examined. Two different loading conditions are considered — axial t .
... Finally, Alexa Fluor (555 or 488)-conjugated avidin was added to visualize antigen-antibody binding sites, using fluorescence Olympus or Nikon Eclipse 80i confocal microscopes (Tokyo, Japan). Cryosections of the cells were also incubated for 60 min with fluorophore-conjugated Ulex europaeus agglutinin (UEA) I lectin (specific for mucous pit cells), Dolichos biflorus agglutinin (DBA) lectin (for parietal cells) or Griffonia simplicifolia (GS)II lectin (for mucous neck cells) (27,28). All lectins were purchased from Sigma (St. Louis, MO, USA). ...
... Such assay will require careful standardization and may even complicate the study, as it is well documented that positive RT-PCR will not always correlate with levels of protein (36) and we have already confirmed mGS cell differentiation into glandular mucous cells using different techniques. Results of both immunohistochemisty and lectin histochemistry demonstrated binding of two very well-characterized biomarkers, anti-TFF2 antibody (29) and GSII lectin (27,28). It is also known that gastric stem cell differentiation into a glandular mucous cells involves increase in cell size due to development of the machinery necessary for production of secretory granules (36). ...
Objectives
To generate various polycaprolactone (PCL) scaffolds and test their suitability for growth and differentiation of immortalized mouse gastric stem (mGS) cells.Materials and methodsNon-porous, microporous and three-dimensional electrospun microfibrous PCL scaffolds were prepared and characterized for culture of mGS cells. First, growth of mGS cells was compared on these different scaffolds after 3 days culture, using viability assay and microscopy. Secondly, growth pattern of the cells on microfibrous scaffolds was studied after 3, 6, 9 and 12 days culture using DNA PicoGreen assay and scanning electron microscopy. Thirdly, differentiation of the cells grown on microfibrous scaffolds for 3 and 9 days was analysed using lectin/immunohistochemistry.ResultsThe mGS cells grew preferentially on microfibrous scaffolds. From 3 to 6 days, there was increase in cell number, followed by reduction by days 9 and 12. To test whether the reduction in cell number was associated with cell differentiation, cryosections of cell-containing scaffolds cultured for 3 and 9 days were probed with gastric epithelial cell differentiation markers. On day 3, none of the markers examined bound to the cells. However by day 9, approximately, 50% of them bound to N-acetyl-d-glucosamine-specific lectin and anti-trefoil factor 2 antibodies, indicating their differentiation into glandular mucus-secreting cells.Conclusions
Microfibrous PCL scaffolds supported growth and differentiation of mGS cells into mucus-secreting cells. These data will help lay groundwork for future experiments to explore use of gastric stem cells and PCL scaffolds in stomach tissue engineering.
... To label dividing progenitor/stem cells in the S phase of the cell cycle, some of the control and treated mice were injected with bromodeoxyuridine (BrdU, 120 mg/kg body weight) 1 hour before sacrifice. Three regions (stomach, small intestine and colon) of the gastrointestinal tract of control and treated mice were processed for histological examination and immunohistochemical analysis as previously mentioned [20]. The proliferative capability and location of these progenitor/stem cells in the gastrointestinal epithelium were previously characterized and well established [20,22]. ...
... Three regions (stomach, small intestine and colon) of the gastrointestinal tract of control and treated mice were processed for histological examination and immunohistochemical analysis as previously mentioned [20]. The proliferative capability and location of these progenitor/stem cells in the gastrointestinal epithelium were previously characterized and well established [20,22]. Tissues were immediately fixed overnight in Bouin solution. ...
Background: Pistacia lentiscus (Anacardiaceae) is a flowering plant traditionally used in the treatment of various skin, respiratory, and gastrointestinal disorders. The aim of this study was to assess whether Pistacia lentiscus oil has any short term toxic effects in vivo and in vitro. Methods: Pistacia lentiscus oil (100µl) was administered orally into mice for 5 days. Results: Measurements of body weight did not show any weight loss. Serum concentration of LDH did not show any significant statistical difference when compared to control mice. Similarly, blood, kidney or liver function tests showed no toxicity with Pistacia lentiscus oil when compared to the control group. Examination of gastrointestinal tissues sections revealed similar structural features with no difference in cell proliferation. In this context, pharmacological dilutions of Pistacia lentiscus oil (10(-6) - 10(-3)) did not affect the viability (cell death and proliferation) of mouse gastric stem cells, human colorectal cancer cells HT29, human hepatoma cells HepG2. However, it appears that at the dose and time point studied, Pistacia lentiscus oil treatment has targeted various cytochrome P450s and has specifically inhibited the activities and the expression of CYP2E1, CYP3A4, CYP1A1 and CYP1A2 differentially in different tissues. Our results also demonstrate that there is no appreciable effect of Pistacia lentiscus oil on the GSH-dependent redox homoeostasis and detoxification mechanism in the tissues. Conclusion: These data suggest a good safety profile of short term oral use of Pistacia lentiscus oil as a monotherapy in the treatment of various skin, respiratory, and gastrointestinal disorders. However, due to its inhibitory effect of various cytochrome P450s and mainly CYP3A4, this might have implications on the bioavailability and metabolism of drugs taken in combination with Pistacia lentiscus oil. More attention is needed when Pistacia lentiscus oil is intended to be uses in combination with other pharmacological agents in order to avoid potential drug-drug interaction leading to toxicity. This study will help in safer use of Pistacia lentiscus oil for therapeutic purpose. © 2014 S. Karger AG, Basel.
... Interestingly, chief cells whose half-life is 194 days have been suggested to be derived by transdifferentiation of mucous neck cells, which takes 14 days [6,7]. The generation and differentiation of chief cells are regulated by the basic helix-loop-helix transcription factor Mist1, and retinoic acid [11][12][13]. Parietal cells, generated from gastric stem cellderived preparietal cells, secrete acid and express various genes, including Arf1, Sod2, Cdhr5, Fads1, Calm2, Igfbp2, and Pthlh [8]; they are regulated by sonic hedgehog (Shh), gastrin, and bone morphogenetic protein (BMP) [14][15][16]. ...
The gastric epithelium is continuously regenerated by gastric stem cells, which give rise to various kinds of daughter cells, including parietal cells, chief cells, surface mucous cells, mucous neck cells, and enteroendocrine cells. The self-renewal and differentiation of gastric stem cells need delicate regulation to maintain the normal physiology of the stomach. Recently, it was hypothesized that cancer stem cells drive the cancer growth and metastasis. In contrast to conventional clonal evolution hypothesis, only cancer stem cells can initiate tumor formation, self-renew, and differentiate into various kinds of daughter cells. Because gastric cancer can originate from gastric stem cells and their self-renewal mechanism can be used by gastric cancer stem cells, we review here how critical signaling pathways, including hedgehog, Wnt, Notch, epidermal growth factor, and bone morphogenetic protein signaling, may regulate the self-renewal and differentiation of gastric stem cells and gastric cancer stem cells. In addition, the precancerous change of the gastric epithelium and the status of isolating gastric cancer stem cells from patients are reviewed.
... Briefly, tissue sections were first deparaffinized, hydrated, and incubated with 3% hydrogen peroxide to block endogenous peroxidase. The anti-BrdU antibody used was goat polyclonal [24] . Biotinylated anti-goat immunoglobulin G was used as a secondary antibody which was identified by avidin-peroxidase and then di-aminobenzidine as a coloring agent. ...
To examine the influence of ghrelin on the regenerative potential of gastrointestinal (GI) epithelium.
Damage to GI epithelium was induced in mice by two intravenous injections of doxorubicin (10 and 6 mg/kg). Some of the doxorubicin-treated mice received a continuous subcutaneous infusion of ghrelin (1.25 μg/h) for 10 d via implanted mini-osmotic pumps. To label dividing stem cells in the S-phase of the cell cycle, all mice received a single intraperitoneal injection of 5'-bromo-2'-deoxyuridine (BrdU) one hour before sacrifice. The stomach along with the duodenum were then removed and processed for histological examination and immunohistochemistry using anti-BrdU antibody.
The results showed dramatic damage to the GI epithelium 3 d after administration of chemotherapy which began to recover by day 10. In ghrelin-treated mice, attenuation of GI mucosal damage was evident in the tissues examined post-chemotherapy. Immunohistochemical analysis showed an increase in the number of BrdU-labeled cells and an alteration in their distribution along the epithelial lining in response to damage by doxorubicin. In mice treated with both doxorubicin and ghrelin, the number of BrdU-labeled cells was reduced when compared with mice treated with doxorubicin alone.
The present study suggests that ghrelin enhances the regenerative potential of the GI epithelium in doxorubicin-treated mice, at least in part, by modulating cell proliferation.
... Cryosections (8 μm thick) were cut and postfixed in methanol for 5 min at −20°C, and washed in PBS (three cycles, 5 min each at room temperature ). Sections were then placed in blocking buffer (1% BSA and 0.3% Triton X-100 in PBS) for 1 h at room temperature and subsequently incubated overnight at 4°C with Fluorescein isothiocyanate (FITC)-conjugated Dolichos biflorus agglutinin (DBA) (Karam et al., 2005). ...
Parietal cells are one of the largest epithelium cells of the mucous membrane of the stomach that secrete hydrochloric acid. To study the function of gastric parietal cells during gastric epithelium homeostasis, we generated a transgenic mouse line, namely, Atp4b-Cre, in which the expression of Cre recombinase was controlled by a 1.0 kb promoter of mouse (-subunit of H(+)-, K(+)-ATPase gene (Atp4b). In order to test the tissue distribution and excision activity of Cre recombinase in vivo, the Atp4b-Cre transgenic mice were bred with the reporter strain ROSA26 and a mouse strain that carries Smad4 conditional alleles (Smad4(Co/Co)). Multiple-tissue PCR of Atp4b-Cre;Smad4(Co/+) mice revealed that the recombination only happened in the stomach. As indicated by LacZ staining, ROSA26;Atp4b-Cre double transgenic mice showed efficient expression of Cre recombinase within the gastric parietal cells. These results showed that this Atp4b-Cre mouse line could be used as a powerful tool to achieve conditional gene knockout in gastric parietal cells.
... wax. A portion of sections (cut at 5 μ m) was stained with haematoxylin and eosin or periodic acid Schiff (PAS) for general histology and mucus staining, respectively. Other sections were used for demonstration of lectin binding and immunohistochemistry using methods as described previously (Falk et al . 1994;Karam et al . 1997Karam et al . , 2004Karam et al . , 2005. Sections were examined with a bright field or fluorescence microscope. ...
... e (FITC)-labelled Grifforia simplifolica II (GSII). These lectins were obtained from Sigma (St. Louis, MO, USA) and have been previously identified and regularly used as reliable lineage-specific markers for pit and neck cells, respectively, of the oxyntic mucosa of developing and adult mice (Falk et al . 1994;Karam et al . 1997Karam et al . , 2004Karam et al . , 2005. Sections of tissue blocks containing both control and TFF1-deficient gastric antral mucosae were deparaffinized, rehydrated, blocked in buffer containing 1% bovine serum albumin, 0.5% Tween-20 in phosphate-buffered saline and finally were incubated for 1 h in UEA-I and GSII lectins at 1 : 600 and 1 : 200 dilutions, respectively. To lab ...
It is not known whether or not epithelial progenitors of the pyloric antrum are involved in gastric carcinogenesis. Normally, these progenitors give rise to two main cell lineages: pit and gland mucous cells. This study was designed to examine the changes that occur in pyloric antral mucous cell lineages and their progenitors during development of gastric adenoma and carcinoma in trefoil factor 1 (TFF1) knockout mice.
Pyloric antral mucosal tissues of TFF1 knockout mice at ages from 3 days to 17 months were processed for histochemical analysis using Ulex europaeus and Grifforia simplifolica lectins as markers for pit and gland mucous cells, respectively. The dividing epithelial progenitors were identified by using immunohistochemical and electron microscopy techniques.
TFF1 loss was associated with amplification of both mucus-secreting pit and gland cells. Both lectins examined bound not only to mature mucous cells, but also to most of epithelial progenitors which gradually amplified with age and frequently were seen in mitosis. Analysis of 12- to 17-month-old TFF1-deficient stomachs revealed occasional groups of poorly differentiated mucosal cells with features similar to those of epithelial progenitors (or stem cells), in the basal portion of the antral mucosa. These cells eventually invaded the muscularis mucosa while maintaining some capacity to differentiate.
This study shows that the progenitors of pit and gland mucous cells contribute to gastric carcinogenesis in the pyloric antrum of TFF1 knockout mice, strongly supporting the concept of stem cell origin of cancer.
... Retinoids have long been known to modulate proliferation and differentiation of various renewing epithelia, and the expression of their receptors has been demonstrated in the gastric mucosa. Karam et al. (2005) recently examined the effects of retinoic acid on progenitor cell proliferation and cell lineage formation in the mouse stomach. They found that retinoic acid enhances the proliferation of gastric epithelial progenitors. ...
It has been suggested that cancer stem cells population within the solid tumor with indefinite proliferation potential drives the growth and metastasis of cancer. In literature, these malignant stem cells also named Cancer initiating cells. Cancer stem cells exhibit low rate of division and proliferation in their niche that help them to avoid chemotherapy and radiation. Epithelial cancers are believed to originate from transformation of tissue stem cells. Bone marrow-derived cells, which are frequently recruited to sites of tissue injury and inflammation, might also represent a potential source of malignancy in the gastrointestinal tract. Pancreatic cancer is one of most common cause of cancer-related death. Pancreatic cancer stem cells have been characterized recently through serial transplantation of human pancreatic cancer cells. The phenotype of Pancreatic cancer stem cells has been defined as CD24(+)CD44(+)CD326 (ESA)(+). CD133 antigen has been also suggested as a potential marker for cancer stem cell in gastrointestinal tract but recently there is also debate in this regard. More recently, other cancer stem cells in gastrointestinal tract, such as colon cancer stem cells, liver cancer stem cells, have been also characterized in their phenotype. These advances clearly will bring the new strategy in cancer treatment and control in the gastrointestinal tract. In this review, the author will discuss the current status and progress about cancer stem cell research in gastrointestinal tract and liver.
