Oral infection with enteropathogenic Escherichia coli triggers immune response and intestinal histological alterations in mice selected for their minimal acute inflammatory responses.

Enteropathogenic Escherichia coli (EPEC), a leading cause of infant diarrhea, is an important public health problem in Brazil and other developing countries. In vitro assays of bacterial adhesion to cultured cells are important tools for studying bacterial pathogenicity but do not reproduce all the events that occur in natural infections. In this study, the effects of oral infection with EPEC on mice selected for their minimal acute inflammatory response (AIR min) were evaluated. Mice were orally infected with EPEC and variations in body weight, bacterial shedding and antibody production observed. The infected animals developed seric and secretory anti-EPEC antibodies; however, neither mortality nor diarrhea was observed. Light microscopy of their intestines demonstrated histological modifications that were not present in controls. However, electron microscopy did not show bacteria attached to the intestinal epithelia to form attaching and effacing lesions, characteristic of EPEC in humans. The bacteria were detected in Peyer's patches and intestinal contents up to 5 hr post-infection. When human anti-EPEC secretory immunoglobulin A or avian immunoglobulin Y antibodies were administered to infected animals, they developed minor histological alterations compared with non-treated animals. In summary, it was found that EPEC triggers immune responses and intestinal histological alterations but does not produce evidence of diarrheal disease in mice infected by the oral route. This study of EPEC experimental infection provides a better understanding of the effects of antibodies on bacterial infections and may provide a suitable model for the design and testing of immunobiological products for active or passive immunization.

Protein restriction inhibits gastric cell proliferation during rat postnatal growth in parallel to ghrelin changes.

OBJECTIVE: Gastric development depends directly on the proliferation and differentiation of epithelial cells, and these processes are controlled by multiple elements, such as diet, hormones, and growth factors. Protein restriction affects gastrointestinal functions, but its effects on gastric growth are not fully understood. METHODS: The present study evaluated cell proliferation in the gastric epithelia of rats subjected to protein restriction since gestation. Because ghrelin is increasingly expressed from the fetal to the weaning stages and might be part of growth regulation, its distribution in the stomach of rats was investigated at 14, 30, and 50 d old. RESULTS: Although the protein restriction at 8% increased the intake of food and body weight, the body mass was lower (P < 0.05). The stomach and intestine were also smaller but increased proportionately throughout treatment. Cell proliferation was estimated through DNA synthesis and metaphase indices, and lower rates (P < 0.05) were detected at the different ages. The inhibition was concomitant with a larger number of ghrelin-immunolabeled cells at 30 and 50 d postnatally. CONCLUSION: Protein restriction impairs cell proliferation in the gastric epithelium, and a ghrelin upsurge under this condition is parallel to lower gastric and body growth rates.

Fasting differentially regulates plasma corticosterone-binding globulin, glucocorticoid receptor, and cell cycle in the gastric mucosa of pups and adult rats.

The nutritional status influences gastric growth, and interestingly, whereas cell proliferation is stimulated by fasting in suckling rats, it is inhibited in adult animals. Corticosterone takes part in the mechanisms that govern development, and its effects are regulated in particular by corticosterone-binding globulin (CBG) and glucocorticoid receptor (GR). To investigate whether corticosterone activity responds to fasting and how possible changes might control gastric epithelial cell cycle, we evaluated different parameters during the progression of fasting in 18- and 40-day-old rats. Food restriction induced higher corticosterone plasma concentration at both ages, but only in pups did CBG binding increase after short- and long-term treatments. Fasting also increased gastric GR at transcriptional and protein levels, but the effect was more pronounced in 40-day-old animals. Moreover, in pups, GR was observed in the cytoplasm, whereas, in adults, it accumulated in the nucleus after the onset of fasting. Heat shock protein (HSP) 70 and HSP 90 were differentially regulated and might contribute to the stability of GR and to the high cytoplasmic levels in pups and elevated shuttling in adult rats. As for gastric epithelial cell cycle, whereas cyclin D1 and p21 increased during fasting in pups, in adults, cyclin E slowly decreased, concomitant with higher p27. In summary, we demonstrated that corticosterone function is differentially regulated by fasting in 18- and 40-day-old rats, and such variation might attenuate any possible suppressive effects during postnatal development. We suggest that this mechanism could ultimately increase cell proliferation and allow regular gastric growth during adverse nutritional conditions.

Opposite effects of fasting on TGF-beta3 and TbetaRI distribution in the gastric mucosa of suckling and early weanling rats.

OBJECTIVE: Our aim was to evaluate the effects of a dietary regimen (suckling or early weaning) and feeding status (fed or fasted) on the distribution of transforming growth factor-beta3 (TGF-beta3) and TGF receptor-I (TbetaRI) in the gastric epithelium of pups. METHODS: Wistar rats were used. At 15 d, half of the pups were separated from dams and fed with hydrated powered chow. On day 17, suckling and early weanling rats were subjected to fasting (17h). Four different conditions were established: suckling fed and fasted and early weanling fed and fasted. At 18 d stomachs were collected under anesthesia and were fixed in 4% formaldehyde for immunohistochemistry. The number of immunostained epithelial cells per microscopic field was determined for TGF-beta3 and TbetaRI in longitudinal sections from the gastric mucosa. RESULTS: We found that during suckling, fasting reduced the number of immunolabeled cells per field of both molecules when compared with the fed group (P<0.05), whereas in early weaning, food restriction increased TGF-beta3 and TbetaRI distributions (P<0.05). We also observed that TGF-beta3 and TbetaRI were more concentrated in parietal cells in the upper gland in suckling pups, whereas after early weaning these were displaced to parietal and chief cells at the bottom of the gland. CONCLUSION: Suckling and early weaning directly influence TGF-beta3 and TbetaRI distributions in the gastric epithelium in response to fasting, such that early weaning anticipates the effects observed in adult rats. Furthermore, the differential concentrations of TGF-beta3 and TbetaRI indicate that they might be important for cell proliferation events in growth control.

Effects of dietary lipid composition and inulin-type fructans on mineral bioavailability in growing rats.

OBJECTIVE: This study reports the effects of feeding with a combination of inulin-type fructans (ITF) and fish oil (FO) on mineral absorption and bioavailability as part of a semipurified diet offered to rats. METHODS: Male Wistar rats (n = 24) were fed a 15% lipid diet (soybean oil [SO] or a 1:0.3 fish:soybean oil mixture [FSO]) and diets containing the same sources of lipids supplemented with 10% ITF (Raftilose Synergy 1) ad libitum for 15 d. Feces and urine were collected for mineral analyses during the last 5 d of the test period. Fatty acid composition was determined in liver and cecal mucosa homogenates. Liver and bone mineral analyses were performed by atomic absorption spectrophotometry. Bone biomechanical analyses were evaluated by a 3-point bending test. RESULTS: Compared with the controls, ITF-fed rats had enlarged ceca and a significant decrease in cecal content pH (P < 0.001). The apparent mineral absorption was improved in these rats, and this effect was enhanced by dietary combination with FO for all minerals except for magnesium. Addition of ITF to the diet resulted in higher bone mineral content (calcium and zinc) and bone strength, but increased bone mineral content was only statistically significant in FO-fed animals. A decrease in liver iron stores (P = 0.015) was observed in rats fed FO, considering that ITF consumption returned to levels comparable to the SO control group. CONCLUSION: These findings confirm the positive influence of ITF on mineral bioavailability, which was potentiated by addition of FO to the diet.

In vivo effects of TGFbeta1 on the growth of gastric epithelium in suckling rats.

As the content of Transforming Growth Factor-beta (TGFbeta) wanes in the milk of lactating rat, an increase in TGFbeta is observed in the gastric epithelia concomitant with differentiation of the glands upon weaning. Whereas TGFbeta has been shown to inhibit the proliferation of gastrointestinal cells in vitro, its functional significance and mechanisms of action have not been studied in vivo. Therefore, we administered TGFbeta1 (1 ng/g body wt.) to 14-day-old rats in which the gastric epithelium was induced to proliferate by fasting, and determined the involvement of signaling through Smads and the impact on epithelial cell proliferation and apoptosis. After the gavage, we observed the progressive increase of active TGFbeta1 while TbetaRII-receptor remained constant in the gastric mucosa. By immunohistochemistry, we showed Smad2/3 increase at 60 min (p<0.05) and Smad2 phosphorylation/activation and translocation to the nucleus most prominently between 0 and 30 min after treatment (p<0.05). Importantly, TGFbeta1 inhibited cell proliferation (p<0.05), which was estimated by BrDU pulse-labeling 12 h after gavage. Lower proliferation was reflected by increased p27(kip1) at 2 h (p<0.05). Also, TGFbeta1 increased apoptosis as measured by M30 labeling at 60 and 180 min (p<0.001), and by morphological features at 12 h (p<0.05). In addition, we observed higher levels of activated caspase 3 (17 kDa) from 0 to 30 min. Altogether, these data indicate a direct effect of TGFbeta1 signaling through Smads on both inhibiting proliferation, through alteration of cycle proteins, and inducing apoptosis of gastric epithelial cells in vivo. Further, the studies suggest a potential role for both milk and tissue-expressed TGFbeta1 in gastric growth during postnatal development.

Myenteric denervation differentially reduces enteroendocrine serotonin cell population in rats during postnatal development.

The enteric nervous and enteroendocrine systems regulate different processes in the small intestine. Ablation of myenteric plexus with benzalkonium chloride (BAC) stimulates epithelial cell proliferation, whereas endocrine serotonin cells may inhibit the process. To evaluate the connection between the systems and the influence of myenteric plexus on serotoninergic cells in rats during postnatal development, the ileal plexus was partially removed with BAC. Rats were treated at 13 or 21 days and sacrificed after 15 days. The cell bodies of myenteric neurons were stained by beta NADH-diaphorase to detect the extension of denervation. The number of enteroendocrine cells in the ileum was estimated in crypts and villi in paraffin sections immunostained for serotonin. The number of neurons was reduced by 27.6 and 45% in rats treated on the 13th and 21st days, respectively. We tried to establish a correlation of denervation and the serotonin population according to the age of treatment. We observed a reduction of immunolabelled cells in the crypts of rats treated at 13 days, whereas this effect was seen in the villi of rats denervated at 21 days. These results suggest that the enteric nervous system might control the enteroendocrine cell population and this complex mechanism could be correlated to changes in cell proliferation.

Intestinal damage in strongyloidiasis: the imbalance between cell death and proliferation.

Strongyloidiasis is an endemic tropical parasitosis caused by Strongyloides stercoralis that also affects immigrants in nontropical countries. The nematode colonizes the duodenum and upper jejunum, inducing mucosal alterations. Because integrity is essential for a functional barrier, we aimed to study apoptosis and proliferation in the small bowel epithelium infected with S. stercoralis. We evaluated 23 patients and 17 controls. Apoptotic cells were detected by TUNEL and M30 immunolabelling, whereas proliferation was scored by Ki67 immunostaining and mitotic counting. Infection increased apoptotic indices in duodenum and jejunum (P < 0.001). Conversely, it decreased cell proliferation in both segments (P < 0.001). Our results showed that intestinal strongyloidiasis promotes an imbalance between cell death and proliferation. This is the first evidence of disruption of the epithelial kinetics with S. stercoralis infection, though the mechanisms remain unclear. Furthermore, our results support the idea that strongyloidiasis disturbs the mucosal integrity and can compromise the intestinal barrier.

Corticosteroids induce the differential expression of TGFbeta isoforms, receptors and signaling in the gastric mucosa of suckling rats.

Glucocorticoids inhibit the cell proliferation in the gastric epithelium, and induce differentiation, migration and death. The mechanism by which these effects are triggered and controlled is still discussed and can involve the transcription and activation of transforming growth factor beta (TGFbeta). The present study was conducted to evaluate the effect of hydrocortisone short-term treatment on tissue level and distribution of TGFbeta isoforms, receptors and signaling through Smad2/3. To achieve that, 18-day-old rats were injected with hydrocortisone (50 mg/Kg b.wt.) for 0, 1 and 3 h. The stomachs were collected and processed for immunohistochemistry and western blotting. We observed that the treatment for 3 h increased the number of labeled epithelial cells for TGFbeta1 (p < 0.05), decreased the distribution of TGFbeta2 (p < 0.05) and did not alter TGFbeta3, TbetaRI and TbetaRII status. The levels of TGFbeta1 and receptors were checked by western blotting and results corroborate the immunodetection. We also found that phosphorylation of Smad2/3 into Smad2P increased after 3 h (p < 0.05), indicating that the high level TGFbeta1 was active on the cells. We suggest that glucocorticoids differentially regulate the expression of TGFbeta isoforms, receptors and signaling, and so TGFbeta1 might be involved in the inhibitory pathway triggered by the hormone.

Ontogenic expression of TGFbeta 1, 2, and 3 and its receptors in the rat gastric mucosa.

The stomach of the rat undergoes extensive changes during the formation and maturation of gastric glands. The presence of transforming growth factor beta (TGFbeta) in rat milk and in the gastrointestinal tract of pups may suggest its role in this process. The current study evaluated the in vivo dynamic expression and distribution of TGFbeta1, beta2, beta3 and their receptors TbetaRI and TbetaRII in the gastric epithelium of 20-day fetal rats and 1-, 14-, 21-, and 30-day-old pups. Immunohistochemistry was used to detect the proteins, and staining was classified according to intensity and cell type. The results showed that the gastric epithelium expresses TGFbeta isoforms and receptors throughout development. We found that immunoreactivity paralleled the appearance of differentiated cells, such that surface mucous cells were the first to be immunostained and chief cells were the last. The intensity of reactions followed this same pattern, showing that the expression of TGFbeta isoforms spread along the gland with growth. Of interest, the highest apparent activity of TGFbeta was observed from 21 days onward, a period that is concomitant with weaning and maturation of most gastric cell types. In addition, surface mucous cells were strongly labeled at the basal cytoplasm at 14 days, suggesting an interaction with the connective tissue. In conclusion, the dynamic expression of TGFbeta1, beta2, beta3, and TbetaRI and TbetaRII through stomach development suggests significant paracrine and autocrine roles for this growth factor. We propose that temporal and spatial differences may be regulated by dietary changes, which in turn control cell proliferation and differentiation in the gastric epithelium.

Goblet cell number in the ileum of rats denervated during suckling and weaning.

The enteric nervous system plays a role on the stimulation of secretory cells of intestinal epithelia. We have demonstrated that ablation of ENS stimulates epithelial cell proliferation. As goblet cells are important constituents of the epithelial sheet, it is mandatory to investigate separately this cell type. The myenteric plexus of the ileum of rats in postnatal development was partially removed by the serosal application of benzalkonium chloride (BAC). Three groups of animals were used: those where BAC application was at 13 days and sacrifice was at 15 (13/28-day-old) or 23 days after treatment (13/36-day-old), and those where BAC was applied at 21 days and rats were killed 15 days after treatment (21/36-day-old) . The number of goblet cells in the ileum was estimated in sections stained by periodic acid-Schiff (PAS) histochemistry. In the 13/28 and 21/36 groups, the number of goblet cells was significantly higher after BAC treatment. These results suggest that the myenteric denervation may have an acute effect on the number of goblet cell in suckling and weanling rats, probably through submucous plexus.

Goblet cell number in the ileum of rats denervated during suckling and weaning.

The enteric nervous system plays a role on the stimulation of secretory cells of intestinal epithelia. We have demonstrated that ablation of ENS stimulates epithelial cell proliferation. As goblet cells are important constituents of the epithelial sheet, it is mandatory to investigate separately this cell type. The myenteric plexus of the ileum of rats in postnatal development was partially removed by the serosal application of benzalkonium chloride (BAC). Three groups of animals were used: those where BAC application was at 13 days and sacrifice was at 15 (13/28-day-old) or 23 days after treatment (13/36-day-old), and those where BAC was applied at 21 days and rats were killed 15 days after treatment (21/36-day-old) . The number of goblet cells in the ileum was estimated in sections stained by periodic acid-Schiff (PAS) histochemistry. In the 13/28 and 21/36 groups, the number of goblet cells was significantly higher after BAC treatment. These results suggest that the myenteric denervation may have an acute effect on the number of goblet cell in suckling and weanling rats, probably through submucous plexus.

Morphological study of the small intestine of moura pigs (Sus scrofa - Lineaus, 1758) during fetal development

The morphology of the small intestine of 39 fetuses and 13 neonates of Brazilian Moura pigs (Sus scrofa) was studied. Fetuses were collected on the 30th, 58th and 86th day of fetal life. The entire small intestine was removed and divided into proximal and distal regions (30th day), and into duodenum, proximal jejunum, distal jejunum and ileum on the 58th and 86th days and in neonates. On the 30th day, the small intestine was small and fragile and there was no visible delimitation among the three segments. The length and diameter of the intestine increased significantly (p<0.001) from 58 days of gestation to parturition. The length of the small intestine, duodenum, jejunum and ileum increased 2.5, 1.2, 2.6 and 3.0 fold, respectively, whereas the diameter increased 2.7, 2.4, 2.7 and 3.0 fold from 58 days of gestation to parturition. On the 30th day, the immature small intestine consisted of mesenchyme and stratified columnar epithelium. On the 58th day, the mucosa, muscularis circular, muscularis longitudinal and serosa were observed in the three segments of small intestine and there were no crypts in the distal jejunum and ileum. Goblet cells were common in the duodenum and rare in the jejunum and ileum. Brünner`s glands were observed in the submucosa. In 86-day fetuses, the presence of incipient myoblasts indicated that the muscularis mucosae was in formation. Crypts were observed in the three segments of the small intestine. In neonates, the muscularis mucosae was present and Brünner`s gland were more frequent. Peyer`s patches were observed in the ileum. These results show that the temporal development of the small intestine of Moura pigs is similar to that of modern breeds. However, macroscopic findings indicate that Moura fetuses have a longer small intestine and heavier body weight at birth than modern breeds.

Effects of somatostatin, LHRH and TGF-(alpha) on epithelial cell proliferation in fetal stomach maintained in organ culture

An organ culture technique was used to examine the effects of somatostatin, luteinizing hormone releasing hormone (LHRH) and transforming growth factor (TGF-alpha) on epithelial cell proliferation in rat fetal stomach. The explants were obtained from 20-day rat fetuses and were maintained in organ culture for 24 h. Half of the culture dishes were supplemented with 10 percent fetal bovine serum (FBS). Cell proliferation was assessed using the metaphasic index. Light and electron microscopy showed that the explants could be mainteined in good condition, independent of the FBS or hormone treatment. Re-epithelialization occurred at the edges of the fragments. The addition of 10 percent FBS was not advantageous for evaluation cell proliferation in this organ culture system. The low metaphasic index showed that somatostatin and LHRH significantly inhibited cell proliferation after 24 h of treatment. In contrast, TGF-alpha had a mitogenic effect on fetal gastric mucosa and prevented glandular degeneration. These results corroborate our previous studies in vivo and provide direct evidence of the influence of hormonal and growth factors on gastric mucosa during fetal development.