Andrés Uribe

Microflora modulates endocrine cells in the gastrointestinal mucosa of the rat

BACKGROUND/AIMS Gastrointestinal peptides and biogenic monoamines participate in the regulation of gastrointestinal functions. The aim of this study was to examine the influence of the microflora on the distribution of endocrine cells and on the release of gastrointestinal peptides. METHODS A quantitative morphological study using stereological methods was performed in gastrointestinal sections of conventional and germ-free rats. Tissue and plasma concentrations of peptides were measured. RESULTS The total volumes of gastrin- and serotonin-immunoreactive cells were significantly increased in the gastric mucosa of germfree rats (P < 0.05), as well as the total volumes of serotonin- and motilin-immunoreactive cells in the ileum (P < 0.05) and serotonin-immunoreactive cells in the colonic mucosa (P < 0.05). The tissue concentration of somatostatin was significantly higher in the jejunum (P < 0.05) and lower in the ileum of germfree rats than in controls (P < 0.05). Plasma glucagon was significantly increased in germfree rats (P < 0.05). The total volume of the fundic mucosa was enlarged in germfree rats (P < 0.05), whereas the total volume, the mucosal thickness, and the number of crypt cells of the colonic mucosa were significantly reduced in these rats compared with controls (P < 0.05). CONCLUSIONS Our findings suggest that the intraluminal microflora influences the release of biologically active peptides and that it participates in the regulation of gastrointestinal endocrine cells and the epithelial structure.

In Vitro Aging of Helicobacter pylori: Changes in Morphology, Intracellular Composition and Surface Properties

During the conversion from the bacillary into the coccoid form, Helicobacter pylori organisms are known to change extensively. The aim of this study was to determine some of the changes that occur regarding morphology, intracellular composition and surface properties during the aging of bacteria in vitro.

E2 prostaglandins modulate cell proliferation in the small intestinal epithelium of the rat

Groups of Sprague-Dawley rats were administered 1 mg/kg indomethacin subcutaneously, indomethacin subcutaneously plus 200 micrograms/kg oral 15-R-15 methyl-prostaglandin E2 (MePGE2) or oral MePGE2 twice daily for 10 days. The animals were treated with antibiotics to prevent mortality. Two control groups were used: control 1 was given placebo and control 2 was treated with antibiotics. All rats were killed 4 h after injection of a metaphase blocker, and the proliferative activity of the distal small intestine was examined in histological sections by means of the cumulative mitotic index (MI). A reduction in the number of villous cells was observed in the rats given antibiotics (p < 0.05 vs. control 1). The small intestinal villi of the rats treated with indomethacin had fewer cells than those of both control groups (p < 0.05) whereas the crypts contained more cells (p < 0.05) and had a higher MI than those of the controls (p < 0.05 vs. controls 1 and 2). These changes were reverted by the prostaglandin analogue. The number of cells of the small intestinal crypts and the cumulative MI in the rats who received indomethacin and the prostaglandin analogue were similar to controls, and they were significantly lower than the values observed in the animals treated with indomethacin (p < 0.05). The animals treated with the prostaglandin analogue and placebo developed a marked hyperplasia of the small intestinal villi (p < 0.05 vs. both control groups), but the atrophy of the villi induced by indomethacin was not prevented by simultaneous administration of the analogue.(ABSTRACT TRUNCATED AT 250 WORDS)

Interleukin-1, interleukin-8, tumour necrosis factor alpha and interferon gamma stimulate DNA synthesis but have no effect on apoptosis in small-intestinal cell lines.

Objectives Cytokines stimulate lymphocyte cell proliferation and affect cell division in several other cell types. Helicobacter pylori-induced gastritis and coeliac disease are characterized by an increased cell proliferation in association with an increased production of pro-inflammatory cytokines, which could contribute to these cell kinetic changes. Our aim is to examine in vitro whether cytokines usually present in the gastrointestinal mucosa affect DNA synthesis and apoptosis in a rat and a human small-intestinal cell line. Methods IEC-6 and FHs-74 cells were incubated for 24 h with 10−13–10−9 M of tumour necrosis factor alpha (TNF-α), interleukin-1β (IL-1β), interleukin-2 (IL-2), interleukin-6 (IL-6), interleukin-8 (IL-8), transforming growth factor-beta (TGF-β) and interferon gamma (IFN-γ). IEC-6 cells were also incubated with 10−13–10−9 M of interleukin-1α (IL-1α) and 10−8 M of interleukin-1 receptor antagonist (IL-1ra). The cells were labelled with 3H-methyl thymidine for the final 4 hours, and then processed for autoradiography. DNA synthesis was evaluated by the labelling index (LI%). Apoptosis was evaluated in IEC-6 cells by changes in membrane lipid asymmetry using annexin-V binding to externalized phosphatidylserine (flow cytometry) and by estimating the caspase activity. Results TNF-α, IL-1β, IL-8 and IFN-γ significantly and markedly increased the LI, even at low concentrations (P < 0.0001), in both IEC-6 and FHs-74 cells, as did IL-1α in IEC-6 cells. TGF-β significantly reduced the LI in both cell lines (P < 0.0001), whereas IL-2, IL-6 and IL-1ra did not affect DNA synthesis significantly. None of IL-1β, IL-8, TNF-α or IFN-γ affected apoptosis in IEC-6 cells. Conclusion TNF-α, IL-1α, IL-1β, IL-8 and IFN-γ stimulated DNA synthesis in a human and a rat small-intestinal cell line. The cytokines exert their mitogenic action directly on the intestinal cells via specific receptors. Our findings indicate that pro-inflammatory cytokines may participate in the regulation of the gastrointestinal epithelial cell proliferation in health and disease.

Helicobacter pylori infection, ABO blood group, and effect of misoprostol on gastroduodenal mucosa in NSAID-treated patients with rheumatoid arthritis

Our aim was to investigate the effect of misoprostol on NSAID-induced gastroduodenal mucosal damage in patients with rheumatoid arthritis. The study included 40 patients, and it was designed as a double-blind, placebo-controlled trial. Misoprostol significantly reduced the gastroduodenal mucosal lesions found at endoscopy (P<0.05) and prevented the development of ulcers. The cumulative incidence of ulcers at four weeks was 5% in the placebo group and 0% in the misoprostol group. The basal and pentagastrin-stimulated acid output as evaluated after 23 days of treatment with misoprostol was not significantly affected. Forty-one percent of the patients had signs of currentHelicobacter pylori infection, 33% had positive serology only, and 26% had no evidence of infection. Most of the patients with current infection belonged to blood group O (P<0.05). Misoprostol treatment did not affect the occurrence ofHelicobacter pylori or the rheumatic disease activity. It is concluded that the protective actions of misoprostol on the gastroduodenal mucosa of NSAID-treated patients are largely mediated by mechanisms other than inhibition of acid secretion. The relationship among activeHelicobacter pylori infection, blood group O, and peptic ulcer may be helpful to identify a subpopulation of patients taking NSAIDs at risk of developing peptic ulcers.

Lipopolysaccharide of Escherichia coli, polyamines, and acetic acid stimulate cell proliferation in intestinal epithelial cells

SummaryOur aim was to examine whether lipopolysaccharide of Escherichia coli, polyamines of dietetic and/or bacterial origin, and products of the bacterial metabolism influence cell proliferation in epithelial cells from the colon and small intestine. Lipopolysaccharide of Escherichia coli 0111:B4 was incubated with cultures from human colonic mucosa. The mitoses were arrested with Vincristine and the total number of metaphases per crypt was counted. In addition, lipopolysaccharide was incubated with a human colonic epithelial cell line from adenocarcinoma (LS-123 cells) and with a nontransformed small intestinal cell line from germ-free rats (IEC-6 cells) for 24 h. In the last 4 h, the cells were labeled with tritiated thymidine. The cells were incubated with putrescine, cadaverine, and spermidine at 10−11–10−3M and with acetic acid (10−5–10−1M), acetaldehyde (10−10–10−4M) and ammonium chloride (1–20 mM). Lipopolysaccharide of Escherichia coli increased the number of arrested metaphases in human colonic crypts and DNA synthesis in L-123 and IEC-6 cells (P<0.001). All polyamines increased DNA synthesis in the colonic and small intestinal cell lines, the effects being more marked for putrescine (P<0.001). The higher concentrations of acetic acid increased DNA synthesis in both epithelial cell lines (P<0.001). Acetaldehyde slightly decreased DNA synthesis in LS-123 cells at cytotoxic concentrations. Ammonium chloride did not significantly affect DNA synthesis. The final concentration of nonionized ammonia was less than 3%. It is concluded that lipopolysaccharides of Escherichia coli and intraluminal factors derived from microorganisms increase cell proliferation in human colonic crypts and intestinal epithelial cell lines.

Helicobacter pylori Stimulates DNA Synthesis in a Small Intestinal Cell Line in vitro

Background: Helicobacter pylori, which causes gastritis and peptic ulcer, seems to be an important factor in the pathogenesis of gastric cancer and MALT lymphoma. Thus our aim was to examine whether H. pylori influences DNA synthesis in epithelial cells in vitro. Methods: Sonicated and water extracts of H. pylori (cytotoxic strains NCTC 11637, 88-23 and A5, and a noncytotoxic isogenic mutant of A5, A5 vac A) were diluted to a final concentration of 1/1,000, 1/100, 1/50 and 1/10. Water extracts of Escherichia coli were used as reference. IEC-6 cells were incubated during 24 h with fragments of H. pylori or extracts of the concentrations described above. The cells were labeled with 3H-methylthymidine for 4 h and processed for autoradiography. DNA synthesis was evaluated by the labeling index (LI). Results: The LI% of controls was 15.6 ± 5.1%. All the water extracts and sonicated strains of H. pylori increased the LI% in a dose-dependent manner (p < 0.001). The highest concentrations of the sonicated strains tended to reduce the LI%, although these values were still higher than those of the control group. The water extracts of E. coli increased the LI% in a dose-dependent manner (p < 0.0001). Conclusion: H. pylori stimulates DNA synthesis in epithelial cells in vitro, but no association was found with the presence of cytotoxin production. Our results suggest that hitherto unknown components of H. pylori may contribute to the increase in cell proliferation observed in gastritis and to the development of MALT lymphoma and gastric cancer.

Indomethacin inhibits cell proliferation and increases cell losses in rat gastrointestinal epithelium

Gastrointestinal cell proliferation was estimated in histological sections of rats treated with low and high doses of parenteral indomethacin for 3 to 60 days. Mitoses were arrested with vincristine and cells in S phase were labeled with tritiated thymidine. Short-term, low-dose treatments reduced the mitotic activity in the oxyntic and small intestinal epithelium, whereas moderate doses restored the mitotic index and high doses increased the proliferative activity and produced epithelial hyperplasia. Long-term, low-dose treatments increased cell proliferation in the small intestine and reduced the number of villous cells. Indomethacin did not affect the proliferative response elicited by refeeding in the oxyntic mucosa, but the simultaneous administration of prostaglandin E2 analog increased the number of arrested mitoses. The turnover of labeled cells was accelerated by indomethacin, particularly in the small intestine. These findings indicate that prostaglandins are regulators of the cell kinetics of the gastrointestinal epithelium but, at the same time, they disclose the presence of trophic mechanisms that are independent of the synthesis of endogenous prostaglandins.

Indomethacin accelerates clearance of labeled cells and increases DNA synthesis in gastrointestinal mucosa of the rat

Sprague-Dawley rats treated with placebo, parenteral indomethacin, or oral prostaglandin E2 for six days were given an intraperitoneal injection of [3H]methyl-thymidine and killed at 45 min and 96 hr after labeling. Treatments were continued, until death. The dpm/DNA index was determined in mucosal scrapings of the stomach, duodenum, and jejunum and used to estimate DNA synthesis (45 min) and the clearance of labeled cells (96 hr). Indomethacin increased the DNA synthesis in both the duodenal and jejunal mucosa (P<0.05). In comparison to the controls, the clearance of labeled cells from the antral, duodenal, and jejunal mucosa was accelerated by indomethacin treatment, whereas the elimination of labeled cells from the antral and jejunal mucosa was slowed by PGE2 treatment (P<0.05). DNA synthesis of the antral mucosa was significantly reduced by PGE2 (P<0.05). The cyclooxygenase blocker did not affect the cell kinetic parameters of the oxyntic mucosa. The plasma levels of somatostatin were significantly higher both in PGE2- and indomethacin-treated rats than in controls (P<0.05). It is concluded that indomethacin treatment increases the cell losses from the epithelial surface, which in turn trigger a compensatory trophic reaction. It is suggested that an important physiological action of endogenous prostaglandins is to regulate the outflow of cells from the superficial zones of the epithelium. Finally, this study disclosed the presence of hitherto unknown regulatory mechanism that promote cell proliferation in the gastrointestinal, mucosa despite inhibition of the synthesis of endogenous prostaglandins.

Enteroscopy as a tool for diagnosing gastrointestinal bleeding requiring blood transfusion.

Iron-deficiency anemia secondary to gastrointestinal blood loss is a common cause of hospitalization. In many cases, the bleeding site cannot be defined despite thorough routine examination of the gastrointestinal tract. The aim of this study was to evaluate push enteroscopy as a diagnostic tool in patients with severe anemia, secondary to recurrent gastrointestinal bleeding, that required management by transfusion. Thirty-five consecutive push enteroscopy investigations were performed in 1998 and 1999 on 25 patients (15 men, 10 women). Mean age was 57 ± 16 years (range, 33–83). All patients had received blood transfusions because of pronounced anemia secondary to gastrointestinal bleeding. Before push enteroscopy, all patients had been investigated with esophagogastroduodenoscopy, colonoscopy, and small-bowel radiography using the double contrast technique; no bleeding site was found. In addition, 10 of 25 patients had been investigated beforehand with 99mTc-labelled red blood cell scintigraphy, and 5 of 25 with scintigraphy for Meckel diverticulum. Two patients were also investigated with angiography before the push enteroscopy, and in six patients an additional total intraoperative enteroscopy was performed, preceded by a new colonoscopy, esophagogastroduodenoscopy, and push enteroscopy. A bleeding site was disclosed in 15 of 25 (60%) patients. In 7 of 25 patients (28%) the bleeding site was found in the stomach or esophagus, even though the patients had undergone one or two esophagogastroduodenoscopies earlier with normal findings. Total intraoperative enteroscopy identified a bleeding site in four of six (67%) patients studied. Two patients had bleeding hemangiomas that were resected surgically. Two patients had small intestinal adenomas, one with adenocarcinoma in situ. Push enteroscopy performed with an overtube inserted under fluoroscopic guidance is an important diagnostic tool in patients in whom conventional examinations do not disclose bleeding sites. Interestingly, 28% of patients had bleeding within reach of the gastroscope, indicating that a new upper endoscopy should be recommended before push enteroscopy is performed. When no positive findings are seen on push enteroscopy and the patient is affected by severe, recurrent iron-deficiency anemia, total intraoperative enteroscopy should be considered.