Sherif M. Karam

Defining Epithelial Cell Progenitors in the Human Oxyntic Mucosa

In the human stomach, the oxyntic epithelium includes numerous tubular invaginations consisting of short pits opening into long glands. The pit is lined by pit cells, whereas the gland is composed of three regions: the base, containing zymogenic cells; the neck, containing neck cells; and the isthmus, composed of little known immature cells and of parietal cells, which are also scattered through the neck and base. The aim of this study was to examine the ultrastructure of the immature cells and to determine their relation to mature cells. To do so, normal oxyntic mucosal biopsies from subjects ranging from 20–43 years old were fixed in aldehydes and postfixed in reduced osmium for electron microscopy and morphometric analysis. The immature cells were sorted out into four classes, whose roles were clarified by comparison with the thoroughly investigated mouse oxyntic epithelium. The first class was composed of the least differentiated immature cells, which were rare and characterized by minute, dense, or cored secretory granules and were accordingly named mini‐granule cells. Their function was not clarified. The second class consisted of pre‐pit cells, which were characterized by few dense mucous granules and give rise to pit cells that ascend the pit wall and, after reaching the luminal surface, die or are extruded. Both pre‐pit and pit cells underwent continuous renewal and, therefore, together constituted a renewal system referred to as pit cell lineage. The third class, or pre‐neck cells, characterized by cored secretory granules, give rise to neck cells that descend toward the base region and differentiate further into pre‐zymogenic cells, which finally become zymogenic cells. The latter eventually degenerate and die. Thus pre‐neck cells and their progeny constitute a renewing system, designated zymogenic cell lineage. The fourth class, or pre‐parietal cells, characterized by long microvilli and few tubulovesicles, differentiate into parietal cells that descend along the neck and base regions and eventually degenerate and die. Pre‐parietal and parietal cells represent a renewing system referred to as parietal cell lineage. While the origin of the last three classes of progenitor cells has not been elucidated, it is likely that they arise either from an unidentified multipotential stem cell, possibly the mini‐granule cell itself, or from the mitotic activity of pre‐pit and pre‐neck cells. In conclusion, the human oxyntic epithelium is composed of continually renewing cells organized in distinct cell lineages.

Slow wave propagation and plasticity of interstitial cells of Cajal in the small intestine of diabetic rats

The number of myenteric interstitial cells of Cajal (ICC‐MY), responsible for the generation and propagation of the slow wave in the small intestine, has been shown to decrease in diabetes, suggesting impairment of slow‐wave (SW) propagation and related motility. To date, however, this expected decrease in SW propagation has neither been recorded nor analysed. Eleven rats were treated with streptozotocin and housed in pairs with 11 age‐matched control animals. After 3 or 7 months, segments of duodenum, jejunum and ileum were isolated and divided into two parts. One part was processed for immediate freezing, cryosectioning and immunoprobing using anti‐c‐Kit antibody to quantify ICC‐MY. The second part was superfused in a tissue bath, and SW propagation was recorded with 121 extracellular electrodes. In addition, a cellular automaton was developed to study the effects of increasing the number of inactive cells on overall propagation. The number of ICC‐MY was significantly reduced after 3 months of diabetes, but rebounded to control levels after 7 months of diabetes. Slow‐wave frequencies, velocities and extracellular amplitudes were unchanged at any stage of diabetes. The cellular automaton showed that SW velocity was not linearly related to the number of inactive cells. The depletion of ICC‐MY is not as severe as is often assumed and in fact may rebound after some time. In addition, at least in the streptozotocin model, the initial reduction in ICC‐MY is not enough to affect SW propagation. Diabetic intestinal dysfunction may therefore be more affected by impairments of other systems, such as the enteric system or the muscle cells.

Functional reentry and circus movement arrhythmias in the small intestine of normal and diabetic rats

In a few recent studies, the presence of arrhythmias based on reentry and circus movement of the slow wave have been shown to occur in normal and diseased stomachs. To date, however, reentry has not been demonstrated before in any other part of the gastrointestinal system. No animals had to be killed for this study. Use was made of materials obtained during the course of another study in which 11 rats were treated with streptozotocin and housed with age-matched controls. After 3 and 7 mo, segments of duodenum, jejunum, and ileum were isolated and positioned in a tissue bath. Slow wave propagation was recorded with 121 extracellular electrodes. After the experiment, the propagation of the slow waves was reconstructed. In 10 of a total of 66 intestinal segments (15%), a circus movement of the slow wave was detected. These reentries were seen in control (n = 2) as well as in 3-mo (n = 2) and 7-mo (n = 6) diabetic rats. Local conduction velocities and beat-to-beat intervals during the reentries were measured (0.42 ± 0.15 and 3.03 ± 0.67 cm/s, respectively) leading to a wavelength of 1.3 ± 0.5 cm and a circuit diameter of 4.1 ± 1.5 mm. This is the first demonstration of a reentrant arrhythmia in the small intestine of control and diabetic rats. Calculations of the size of the circuits indicate that they are small enough to fit inside the intestinal wall. Extrapolation based on measured velocities and rates indicate that reentrant arrhythmias are also possible in the distal small intestine of larger animals including humans.

Characterization of Breast Cancer Progression in the Rat

The incidence of breast cancer is continuously increasing worldwide. This increasing trend is attributed partly to the fact that a considerable number of cases are related to environmental factors and partly to the little information available on the early changes that occur during mammary gland carcinogenesis. To characterize some of these early cellular changes, breast cancer was induced in female rats using a single intragastric dose of the environmental carcinogen 7,12‐dimethylbenz[a]anthracene (DMBA; 80 mg/kg body weight). Mammary gland tissues of control and DMBA‐treated rats were processed for routine histopathological examination and immunohistochemical analysis using an antibody specific for the proliferating cell nuclear antigen (PCNA). Microscopic examination of all mammary glands of DMBA‐treated rats revealed a wide range of preneoplastic stages in addition to the well‐characterized benign and malignant tumors that developed. The first stage was characterized by slightly dilated terminal ducts with accumulation of dead cells. This was designated the stage of cell death. Then, stages of hyperplasia, dysplasia, and carcinoma in situ followed. Immunohistochemical localization of PCNA in these preneoplastic lesions revealed an initial decrease followed by a gradual increase in the labeling index of PCNA. In conclusion, the DMBA‐treated rats provide a useful model to dissect the early changes that occur during the multistep process of mammary gland carcinogenesis.

Cellular Origin of Gastric Cancer

Gastric cancer is the second leading cause of cancer deaths worldwide. Although the link between Helicobacter pylori infection and gastric cancer is well established, little is known about the early development and detection of this malignant disease. Cancer is the disease of epithelia and recently, it has been suggested that some cancers originate in adult stem cells. Advances have been made in identifying the gastric epithelial stem cells and their immediate descendents, which act as progenitors giving rise to mucus‐, acid‐, pepsinogen‐, and hormone‐secreting cell lineages. Analyses of some genetically manipulated animal models in which the proliferation and differentiation program of the gastric stem/progenitor cells was altered by different approaches have provided some clues to the cellular origin of gastric cancer. Despite the challenges and the similarity between gastric epithelial progenitors and their differentiation program in mice and humans, it remains to be determined whether observations made in genetically engineered mice are also applicable to humans.

Retinoic acid stimulates the dynamics of mouse gastric epithelial progenitors.

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 mucosa. The aim of this study was to examine the effects of retinoic acid on progenitor cell proliferation and cell lineage formation in the mouse stomach. By using subcutaneously inserted osmotic pumps, mice were continuously infused with all‐trans retinoic acid (5 mg/kg per day) for 3 days. To label S‐phase cells and their progeny, bromodeoxyuridine was administered for different time intervals. Analysis of gastric mucosal tissues of retinoic acid–treated mice revealed a significant increase in the number of S‐phase progenitor cells and an enhancement in the production of their progeny. The life span of pit cells was reduced, and their apoptosis became apparent at the luminal surface. Immunofluoresence probing of pit, parietal and enteroendocrine cell lineages in control and retinoic acid–treated mice showed no significant change in their labeling pattern. However, there was an increase in the labeled gland area of zymogenic cells. In conclusion, 3‐day treatment of retinoic acid enhances the proliferation of gastric epithelial progenitors and the dynamics of their progeny.

Potential role of probiotics in the management of gastric ulcer (Review)

Gastric ulcer is one of the most common chronic gastrointestinal diseases characterized by a significant defect in the mucosal barrier. Helicobacter pylori (H. pylori) infection and the frequent long-term use of non-steroidal anti-inflammatory drugs are major factors involved in gastric ulcer development. Acid inhibitors and antibiotics are commonly used to treat gastric ulcer. However, in the last few decades, the accumulating evidence for resistance to antibiotics and the side effects of antibiotics and acid inhibitors have drawn attention to the possible use of probiotics in the prevention and treatment of gastric ulcer. Probiotics are live microorganisms that when administered in adequate amounts confer health benefits on the host. Currently, the available experimental and clinical studies indicate that probiotics are promising for future applications in the management of gastric ulcers. This review aims to provide an overview of the general health benefits of probiotics on various systemic and gastrointestinal disorders with a special focus on gastric ulcer and the involved cellular and molecular mechanisms: i) Protection of gastric mucosal barrier; ii) upregulation of prostaglandins, mucus, growth factors and anti-inflammatory cytokines; iii) increased cell proliferation to apoptosis ratio; and iv) induction of angiogenesis. Finally, some of the available data on the possible use of probiotics in H. pylori eradication are discussed.

Short-term effects of oral administration of Pistacia lentiscus oil on tissue-specific toxicity and drug metabolizing enzymes in mice.

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.

Expression of retinoid receptors in multiple cell lineages in the gastric mucosae of mice and humans

Background and Aim:  In mice and humans, the gastric epithelial progenitors undergo proliferation and bipolar migration from the isthmus associated with their differentiation into mucus‐, acid‐ and pepsinogen‐secreting cell lineages. Little is known about factors that control the dynamics of these isthmal progenitor cells. Retinoids have long been known as chemopreventive agents against gastric mucosal damage and carcinogenesis. The aim of the present study was to examine the cellular localization of the various retinoid receptors proteins (RAR and RXR) in the gastric epithelium of mice and humans.

NOTCH3 is expressed in human apical papilla and in subpopulations of stem cells isolated from the tissue

NOTCH plays a role in regulating stem cell function and fate decision. It is involved in tooth development and injury repair. Information regarding NOTCH expression in human dental root apical papilla (AP) and its residing stem cells (SCAP) is limited. Here we investigated the expression of NOTCH3, its ligand JAG1, and mesenchymal stem cell markers CD146 and STRO-1 in the AP or in the primary cultures of SCAP isolated from AP. Our in situ immunostaining showed that in the AP NOTCH3 and CD146 were co-expressed and associated with blood vessels having NOTCH3 located more peripherally. In cultured SCAP, NOTCH3 and JAG1 were co-expressed. Flow cytometry analysis showed that 7%, 16% and 98% of the isolated SCAP were positive for NOTCH3, STRO-1 and CD146, respectively with a rare 1.5% subpopulation of SCAP co-expressing all three markers. The expression level of NOTCH3 reduced when SCAP underwent osteogenic differentiation. Our findings are the first step towards defining the regulatory role of NOTCH3 in SCAP fate decision.