Leanna C. Read

Transforming Growth Factor-β Levels in Maternal Milk and Expression in Postnatal Rat Duodenum and Ileum

After birth, the gastrointestinal tract of the neonate is exposed to food and bacterial and environmental antigens. Maternal milk components may play a role in regulation of mucosal immune activity to luminal antigens. In this study we determine the ontogeny of transforming growth factor (TGF)-β1-producing cells in the rat pup small intestine and assess maternal milk concentrations of TGF-β. Intestinal tissue samples of duodenum and ileum were collected, processed, and stained for TGF-β1, and in situ hybridization for TGF-β1 mRNA was also performed on the duodenum. TGF-β levels in milk were assayed by ELISA. TGF-β2 levels in milk were high at d 6, and declined thereafter at d 10 and 19. TGF-β1 was not detected. In contrast, the cell number and intensity of staining of TGF-β1 peptide in the small intestine was low in 3- and 10-d-old rats and increased markedly by 19 d of life. In the duodenum mRNA levels mirrored this trend. TGF-β1 expression in the lamina propria was absent before d 19, and increased progressively over time. Maternal milk TGF-β2 levels are high in early milk and decrease during the weaning period. In contrast, endogenous TGF-β production in the small intestine increases during the weaning period.

Des(1–3)IGF-I: a truncated form of insulin-like growth factor-I

Des(1-3)IGF-I, a truncated variant of human IGF-I with the tripeptide Gly-Pro-Glu absent from the N-terminus, has been isolated from bovine colostrum, human brain and porcine uterus. This protein probably results from post-translational cleavage of IGF-I. Des(1-3)IGF-I generally is about 10-fold more potent than IGF-I at stimulating hypertrophy and proliferation of cultured cells, a consequence of much reduced binding to IGF-binding proteins, in turn caused by the absence of the glutamate at position 3. The increased potency is retained in part when the variant is administered in vivo, with selective anabolic effects particularly evident in gut tissues. Clinical opportunities for des(1-3)IGF-I have not yet been evaluated, but could apply in catabolic states as well as for the treatment of inflammatory bowel diseases.

Temporal changes in TFF3 expression and jejunal morphology during methotrexate-induced damage and repair

Trefoil factor TFF3 has been implicated in intestinal protection and repair. This study investigated the spatiotemporal relationship between TFF3 expression and morphological changes during intestinal damage and repair in a rat model of methotrexate-induced small intestinal mucositis. Intestinal tissues from rats with mucositis were collected daily for 10 days. Mucosal damage was characterized by an initial decrease in cell proliferation resulting in crypt loss, villus atrophy, and depletion of goblet cells, followed by hyperproliferation that lead to crypt and villus regeneration and mucous cell repopulation. TFF3 mRNA levels increased marginally during histological damage, and the cell population expressing TFF3 mRNA expanded from the usual goblet cells to include some nongoblet epithelial cells before goblet cell repopulation. TFF3 peptide, however, was depleted during histological damage and normalized during repair, mirroring the disappearance and repopulation of goblet cells. Although there is no temporal relationship between TFF3 levels and crypt hyperproliferation, confirming the nonmitogenic nature of TFF3, the coincidental normalization of TFF3 peptide with repopulation of goblet cells and mucin production after proliferative overshoot suggests that TFF3 may play a role in the remodeling phase of repair.Trefoil factor TFF3 has been implicated in intestinal protection and repair. This study investigated the spatiotemporal relationship between TFF3 expression and morphological changes during intestinal damage and repair in a rat model of methotrexate-induced small intestinal mucositis. Intestinal tissues from rats with mucositis were collected daily for 10 days. Mucosal damage was characterized by an initial decrease in cell proliferation resulting in crypt loss, villus atrophy, and depletion of goblet cells, followed by hyperproliferation that lead to crypt and villus regeneration and mucous cell repopulation. TFF3 mRNA levels increased marginally during histological damage, and the cell population expressing TFF3 mRNA expanded from the usual goblet cells to include some nongoblet epithelial cells before goblet cell repopulation. TFF3 peptide, however, was depleted during histological damage and normalized during repair, mirroring the disappearance and repopulation of goblet cells. Although there is no temporal relationship between TFF3 levels and crypt hyperproliferation, confirming the nonmitogenic nature of TFF3, the coincidental normalization of TFF3 peptide with repopulation of goblet cells and mucin production after proliferative overshoot suggests that TFF3 may play a role in the remodeling phase of repair.

Exposure of oral mucosa to bioactive milk factors reduces severity of chemotherapy-induced mucositis in the hamster

A biologically active extract containing bovine whey proteins, whey growth factor extract-A (WGFE-A) was administered topically to the oral mucosa of hamsters and its ability to prevent and treat chemotherapy-induced oral mucositis investigated. Oral mucositis was induced in Syrian golden hamsters through a combination treatment of the antimetabolite chemotherapy drug 5-fluorouracil (5-FU), and mild abrasion of the cheek pouch. WGFE-A administered to the oral mucosa via hydrogel and liquid treatments, pre and concurrent to 5-FU therapy, resulted in significantly reduced mucosal ulceration. The protective effect was dose dependent with greatest benefit from WGFE-A doses applied at 4.2 mg/ml gel and 14 mg/ml mouthwash (P<0.01). The protective activity of WGFE-A also appeared related to mode of delivery. Administration of WGFE-A from an alternate vehicle Orabase(R) did not alleviate mucositis compared to WGFE-A applied in hydrogel. When administered continuously after the chemotherapy schedule, WGFE-A failed to reduce ulcer area when applied over a 12-day period. In a separate study, cell cycle staining indicated that cheek pouch mucosal epithelial cells pre-exposed to WGFE-A in-vivo showed a reduced rate of proliferation, measured as a 21% reduction in the bromodeoxyuridine (BrdU) cell labelling index (P<0.04). This was consistent with a protective mode of WGFE-A action against anti-metabolites such as 5-FU which target rapidly dividing cells. The results were also consistent with recent in vitro data showing protective properties from WGFE-A administered to epithelial cells given pre/concurrent to chemotherapy exposure. WGFE-A is known to contain mitogens which stimulate cells of mesenchymal origin and inhibit epithelial cell growth in culture. Several WGFE-A constituents are likely to confer protective effects on the cheek mucosa, including anti-proliferative, anti-apoptotic and anti-microbial factors. WGFE-A provides a potentially valuable source of topically delivered proteins for clinical application in preventing severe oral mucositis caused by chemotherapy.

Predisposition to colonic dysplasia is unaffected by continuous administration of insulin-like growth factor-I for twenty weeks in a rat model of chronic inflammatory bowel disease.

Abstract Background Insulin-like growth factor-I (IGF-I) is currently under evaluation for the treatment of a variety of chronic disease conditions. We investigated the safety of long-term IGF-I administration in a rat model of inflammatory bowel disease which predisposes to the development of dysplasia. Methods Chronic consumption of dextran sulphate sodium (DSS) by rats manifests a colitis with dysplastic features. Rats consumed 2% DSS for 4 weeks when pumps were implanted to deliver either vehicle or IGF-I for 15 or 20 weeks while rats continued to consume DSS. Features of colitis and dysplasia were assessed at kill. Results Compared to vehicle, 20 weeks IGF-I significantly increased body weight by 19% and total gut weight by 43%. Colonic crypt depth, proliferative compartment, labelling index, dysplasia, neoplasia and other indices of colitis were not significantly affected. Conclusions Twenty weeks administration of IGF-I to rats induced growth of the intestine but did not affect the severity of experimentally-induced colitis or the incidence or progression of colonic dysplasia.

Use of the 13C-sucrose breath test to assess chemotherapy-induced small intestinal mucositis in the rat

Mucositis is a debilitating side-effect of chemotherapy which affects the mucosa of the gastrointestinal tract, particularly the small intestine. Currently there are no simple, non-invasive methods to detect and monitor small intestinal function and the severity of mucosal damage. Activity of the brush-border enzyme sucrase provides an indicator of small intestinal absorptive function that remains relatively constant throughout life. Measuring 13CO-2 levels in expired breath following ingestion of 13C-sucrose is a non-invasive marker of total intestinal sucrase activity. We evaluated the sucrose breath test (SBT) as an indicator of small intestinal injury and dysfunction, utilizing a rat model of chemotherapy-induced mucositis. SBT results reflected the time-course of damage and repair after methotrexate (MTX) treatment, with damage most severe 72 h after chemotherapy, and repair commencing after 96 h. SBT results correlated significantly with jejunal sucrase activity determined biochemically (r2=0.89; p

Applicability of the Ussing Chamber Technique to Permeability Determinations in Functionally Distinct Regions of the Gastrointestinal Tract in the Rat

Background: Ussing chambers are commonly utilized for in vitro investigations into gastrointestinal permeability. However, their sensitivity and applicability to the small intestine have not been well characterized. Methods: In order to investigate the effects of experimentally induced damage and the relative contribution of the mucosa and muscularis externa layers to transmural permeability in the small intestine, stomach and colon, normal rat intestinal tissues were mounted in Ussing chambers with or without removal of the muscularis externa or mucosal layers. Gastric tissues were damaged in vivo by exposure to indomethacin (100 r mg r kg m 1 ), while ileal tissues were damaged in vitro by 0.4 r M NaCl. Tissue damage was assessed histologically, while permeability parameters included conductance (G), potential difference (PD) and mucosal to serosal flux of horseradish peroxidase (HRP). Results: Damage localized to the tissue edges (edge damage) accounted for 25%-50% of the exposed epithelial length in the ileum, while less than 20% of stomach and colon epithelium was affected by edge damage. In the damaged stomach, a 20% reduction in epithelialization was accompanied by increases in G ( P r < r 0.001) and HRP ( P r < r 0.01) flux. Removal of the muscularis externa did not affect mucosal viability in the undamaged ileum or colon although HRP flux in the colon, but not ileum, was increased ( P r < r 0.01). Removal of the ileal mucosa produced increases in G and HRP flux, while PD was maintained. Conclusion: We conclude that the Ussing chamber technique is suitable for application to studies of gastric and colonic permeability in rats. However, owing to the prevalence and extent of edge damage in the small intestine, we would caution against the use of this technique for permeability studies in this region of the gastrointestinal tract in the rat.BACKGROUND Ussing chambers are commonly utilized for in vitro investigations into gastrointestinal permeability. However, their sensitivity and applicability to the small intestine have not been well characterized. METHODS In order to investigate the effects of experimentally induced damage and the relative contribution of the mucosa and muscularis externa layers to transmural permeability in the small intestine, stomach and colon, normal rat intestinal tissues were mounted in Ussing chambers with or without removal of the muscularis externa or mucosal layers. Gastric tissues were damaged in vivo by exposure to indomethacin (100 mg kg(-1)), while ileal tissues were damaged in vitro by 0.4 M NaCl. Tissue damage was assessed histologically, while permeability parameters included conductance (G), potential difference (PD) and mucosal to serosal flux of horseradish peroxidase (HRP). RESULTS Damage localized to the tissue edges (edge damage) accounted for 25%-50% of the exposed epithelial length in the ileum, while less than 20% of stomach and colon epithelium was affected by edge damage. In the damaged stomach, a 20% reduction in epithelialization was accompanied by increases in G (P < 0.001) and HRP (P < 0.01) flux. Removal of the muscularis externa did not affect mucosal viability in the undamaged ileum or colon although HRP flux in the colon, but not ileum, was increased (P < 0.01). Removal of the ileal mucosa produced increases in G and HRP flux, while PD was maintained. CONCLUSION We conclude that the Ussing chamber technique is suitable for application to studies of gastric and colonic permeability in rats. However, owing to the prevalence and extent of edge damage in the small intestine, we would caution against the use of this technique for permeability studies in this region of the gastrointestinal tract in the rat.

Localization of transforming growth factor-beta receptor types I, II, and III in the postnatal rat small intestine.

Transforming growth factor-β2 (TGF-β2) levels in rat milk are high in early lactation, whereas endogenous TGF-β1 expression in the neonatal gut increases toward midweaning. Three types of transmembrane TGF-β receptors have been identified in mammals. The receptor III (or betaglycan) binds and presents TGF-β1 or β2 to receptor II. Receptor I then interacts with receptor II, forming a signaling receptor complex, and propagates the signal. To determine whether TGF-β receptor expression in the gut is also developmentally regulated, the present study assessed ontogeny of TGF-β receptor expression in the postnatal rat small intestine. Jejunum and ileum tissues from rat pups at d 3, 10, 14, 21, and 28 of age were collected. Cryostat sections were stained with antibodies against TGF-β receptors I, II, and III, and various cell markers by immunofluorescence. In both regions, receptor I staining was seen on apical and basolateral membranes of the villus and crypt epithelium at all ages, and staining on the apical membrane increased with age; receptor II was predominantly expressed in the crypt, and staining on the villi appeared after d 10; receptor III was distributed throughout the mucosa at early ages but diminished from the epithelium postweaning by d 28. T cells, B cells, and dendritic cells in the lamina propria expressed TGF-β receptor III but lacked expression of receptor I and II. The pattern of TGF-β receptor expression changes with age in a manner that may reflect the change in ligand from TGF-β2 (milk-derived) to TGF-β1 (endogenously produced).

Effects of TGF-α gene knockout on epithelial cell kinetics and repair of methotrexate-induced damage in mouse small intestine

While previous studies have indicated that exogenous TGF‐α stimulates epithelial growth, maintenance, and repair of the gut, roles of endogenous TGF‐α are less well‐defined particularly in the small bowel. The current study examined effects of TGF‐α knockout on adult small intestinal epithelial cell proliferation, migration, apoptosis, and damage/repair response after methotrexate treatment. Compared to normal mice, TGF‐α gene knockout did not affect crypt cell production, mitosis position, migration, and apoptosis in non‐injured intestine. RT‐PCR gene expression analysis revealed presence of four out of six TGF‐α related EGF family ligands in the normal intestine, suggesting a possible functional redundancy of the EGF family in maintenance of the intestine. Although TGF‐α gene knockout did not significantly impair the overall mucosal repair in methotrexate‐induced acute damage in the small intestine, it resulted in a higher apoptotic response in the early hours following methotrexate challenge, and a delayed and reduced crypt cell proliferation during repair. Consistently, after methotrexate challenge, intestinal TGF‐α mRNA was found to be markedly upregulated in the early hours and during repair in the wild type, and there were similar profiles in the increased expression of all other ligands (except EGF) between the wild type and knockout intestines. Therefore, despite a possible functional redundancy among the EGF family ligands in the normal small intestine, TGF‐α may play a role in modulating the early apoptotic events and in enhancing the subsequent reparative proliferative response in the methotrexate‐damaged intestine. J. Cell. Physiol. 191: 105–115, 2002.

Subcutaneous but not Intraluminal Epidermal Growth Factor Stimulates Colonic Growth in Normal Adult Rats

Epidermal growth factor (EGF) was administered by chronic subcutaneous or intracolonic infusion into normal adult rats to determine the effect on colonic growth. Subcutaneous infusion of 200 micrograms EGF/kg/day for 7 days increased the cross-sectional mass and protein content of the muscularis and mucosal layers of the proximal colon, with the distal colon showing less response. In the mucosa, subcutaneous EGF induced proportional increments in the number of cells per crypt, and in the number of cells positively labelled for PCNA, while maintaining a normal crypt growth fraction. In contrast, an 8-fold higher dose of EGF administered intraluminally had no effect on colonic mucosal or muscularis growth. This lack of bioactivity was unlikely to reflect rapid luminal degradation as radiolabelled EGF remained stable in the colonic lumen for at least 4 h. The results demonstrate that the normal adult colon is responsive to subcutaneously delivered EGF, particularly the proximal colon, whereas EGF may not be active on the normal colon when presented from the luminal direction.