Tania Marchbank

Human spasmolytic polypeptide is a cytoprotective agent that stimulates cell migration

BACKGROUND/AIMS Gastric epithelium is attacked by acid, pepsin, and ingested agents. When a mucosal lesion occurs, the defect is rapidly closed by cell migration. Because spasmolytic polypeptide is rapidly produced at sites of injury, we postulated that human spasmolytic polypeptide (hSP) was important in mucosal repair. Recombinant hSP was used to test this hypothesis. METHODS The ulcer healing effect of various doses of hSP administered orally and subcutaneously was examined using an indomethacin (20 mg/kg) restraint rat model of gastric damage. Stability of hSP in gastrointestinal juice was determined using size-exclusion chromatography. The effect of hSP on migration of human colonic carcinoma cell lines HT29 and SW480 was determined using collagen gel invasion and wounded monolayer assays. Proliferation was assessed using [3H]thymidine incorporation and toluidine blue staining. RESULTS Infusions of hSP at 25 and 50 micrograms.kg-1.h-1 subcutaneously decreased gastric damage by about 50% (P < 0.01) without changing acid secretion. Oral hSP was ineffective. hSP was stable in gastrointestinal juice. hSP stimulated migration of HT29 cells but did not affect proliferation and had no effect on SW480 cells. CONCLUSIONS hSP may play a key role in the early stages of mucosal repair by stimulating the initial re-epithelialization by cell migration.

Bovine colostrum is a health food supplement which prevents NSAID induced gut damage

BACKGROUND Non-steroidal anti-inflammatory drugs (NSAIDs) are effective for arthritis but cause gastrointestinal injury. Bovine colostrum is a rich source of growth factors and is marketed as a health food supplement. AIMS To examine whether spray dried, defatted colostrum or milk preparations could reduce gastrointestinal injury caused by indomethacin. METHODS Effects of test solutions, administered orally, were examined using an indomethacin restraint rat model of gastric damage and an indomethacin mouse model of small intestinal injury. Effects on migration of the human colonic carcinoma cell line HT-29 and rat small intestinal cell line RIE-1 were assessed using a wounded monolayer assay system (used as an in vitro model of wound repair) and effects on proliferation determined using [3H]thymidine incorporation. RESULTS Pretreatment with 0.5 or 1 ml colostral preparation reduced gastric injury by 30% and 60% respectively in rats. A milk preparation was much less efficacious. Recombinant transforming growth factor β added at a dose similar to that found in the colostrum preparation (12.5 ng/rat), reduced injury by about 60%. Addition of colostrum to drinking water (10% vol/vol) prevented villus shortening in the mouse model of small intestinal injury. Addition of milk preparation was ineffective. Colostrum increased proliferation and cell migration of RIE-1 and HT-29 cells. These effects were mainly due to constituents of the colostrum with molecular weights greater than 30 kDa. CONCLUSIONS Bovine colostrum could provide a novel, inexpensive approach for the prevention and treatment of the injurious effects of NSAIDs on the gut and may also be of value for the treatment of other ulcerative conditions of the bowel.

Epidermal growth factor is digested to smaller, less active forms in acidic gastric juice.

BACKGROUND/AIMS Epidermal growth factor (EGF) is present in gastric juice and has potent mitogenic properties. The stability of EGF in gastric juice under various physiological and pathophysiological conditions was examined. METHODS Recombinant human EGF1-53 was incubated with HCl containing pepsin. We also determined the forms of EGF present in the gastric juice of patients under basal conditions, patients taking the acid suppressant omeprazole, patients with achlorhydria, and volunteers undergoing intragastric neutralization with NaHCO3 (n = 6 per group). Samples were analyzed using mass spectroscopy and/or high-pressure liquid chromatography followed by radioimmunoassay. The effect of acid and pepsin digestion on EGF bioactivity was determined using an in vitro hepatocyte bioassay and an in vivo cytoprotection assay in the rat stomach. RESULTS EGF1-53 was digested to the EGF1-49 and EGF1-46 forms in all samples containing pepsin when the pH was < 4. In gastric juice samples with pH > 4, the proportion of intact EGF increased to about 60%. For both methods of bioassay, intact EGF1-53 was about 3-4 times as potent as acid and pepsin-treated EGF. CONCLUSIONS EGF is produced in the 1-53 form but is rapidly cleaved to smaller, less active forms in acidic gastric juice. In contrast, only a small proportion of the EGF is cleaved if the pH is maintained above 4. This mechanism may be relevant to the healing process of acid suppressants.

Dimerization of human pS2 (TFF1) plays a key role in its protective/healing effects.

Human pS2 (trefoil factor family 1, TFF1), a 60‐amino acid member of the trefoil peptide family, forms dimers via Cys58 and may stimulate gut repair. The effects of dimeric pS2‐TFF1 and monomeric pS2‐TFF1 (Cys58 replaced by Ser58) were compared in models of wound healing. Rats given dimeric pS2‐TFF1 at 25 and 50 μg/kg per h had 50 per cent and 70 per cent reduction in gastric damage induced respectively by indomethacin (20 mg/kg subcutaneously) and restraint (P<0·01). Monomeric pS2‐TFF1, at the same doses, was significantly less effective at reducing injury (about half the amount of protection, P<0·01 vs. same doses of dimeric). The rate of migration of cells at the leading edge of wounded monolayers of the human colonic cell line HT29 was increased by addition of dimeric or monomeric forms of pS2‐TFF1 (0·65–325 μg/ml). Dimeric pS2‐TFF1 had a greater effect than the monomeric form at all doses tested (P<0·05). Cell migration induced by pS2‐TFF1 was blocked by a pS2‐TFF1 antibody, but not by a transforming growth factor β neutralizing antibody. pS2‐TFF1 did not influence cell proliferation as assessed by thymidine incorporation. The increased biological effects of dimeric pS2‐TFF1 might be due to direct interaction of Cys58 with a putative trefoil receptor or, more likely, dimerization of pS2‐TFF1 might stabilize the interaction with its receptor. This may involve a bivalent interaction of residues on the surfaces of the two trefoil domains.

The trefoil peptide TFF1 inhibits the growth of the human gastric adenocarcinoma cell line AGS

TFF1 is a 60‐amino acid peptide produced in normal gastric mucosa which forms dimers spontaneously. Tumours of patients with gastric cancer usually have reduced TFF1 levels and disruption of the TFF1 gene causes animals to develop gastric adenomas and carcinomas. The effect of normal sequence human recombinant TFF1 and an analogue (Cys58→Ser58), which is unable to dimerize, on the proliferation and morphology of the human gastric adenocarcinoma cell line AGS was therefore investigated. Proliferation, assessed by total cell number and [methyl‐3H]thymidine incorporation, was reduced by dimeric TFF1 in a dose‐dependent manner. Monomeric TFF1 also reduced proliferation but was less potent than the dimeric form. It is concluded that TFF1 may be an important controller of gastric cell proliferation, that dimerization of TFF1 is important in this effect, and that the reduced levels of TFF1 seen in gastric cancer may be of clinical relevance. Copyright

Human pancreatic secretory trypsin inhibitor : Distribution, actions and possible role in mucosal integrity and repair

Pancreatic secretory trypsin inhibitor is a potent protease inhibitor which was originally identified in the pancreas. It has subsequently been shown to be present in mucus-secreting cells throughout the gastrointestinal tract and also in the kidney, lung and breast. Its major roles are likely to be to prevent premature activation of pancreatic proteases and to decrease the rate of mucus digestion by luminal proteases within the stomach and colon. In addition, PSTI increases the proliferation of a variety of cell lines and stimulates cell migration, possibly acting via the EGF receptor. These findings suggest that PSTI may also be involved in both the early and late phases of the healing response following injury. Further studies including the production of transgenic overexpression and knockout models should help elucidate the physiological function of this peptide.

Reparative properties of a commercial fish protein hydrolysate preparation

Background: A partially hydrolysed and dried product of pacific whiting fish is currently marketed as a health food supplement to support “intestinal health”. However, there has been only limited scientific study regarding its true biological activity. Aims: We therefore tested its efficacy in a variety of models of epithelial injury and repair. Methods: Effects on proliferation were determined using [3H] thymidine incorporation into epithelial rat intestinal RIE-1 and human colonic HT29 cells. Effects on restitution (cell migration) were analysed using wounded HT29 monolayers and its ability to influence gastric injury analysed using a rat indomethacin restraint model. Partial characterisation of bioactive agents was performed using mass spectroscopy, high pressure liquid chromatography, and gas chromatography. Results: Both cell proliferation and cell migration were increased by about threefold when added at 1 mg/ml (p<0.01). Gastric injury was reduced by 59% when gavaged at 25 mg/ml (p<0.05), results similar to using the potent cytoprotective agent epidermal growth factor at 12.5 μg/ml. The vast majority of biological activity was soluble in ethanol, with glutamine in its single, di-, and tripeptide forms probably accounting for approximately 40% of the total bioactivity seen. Fatty acid constituents may also have contributed to cell migratory activity. Conclusions: Fish protein hydrolysate possesses biological activity when analysed in a variety of models of injury and repair and could provide a novel inexpensive approach for the prevention and treatment of the injurious effects of non-steroidal anti-inflammatory drugs and other ulcerative conditions of the bowel. Further studies appear justified.

Zinc carnosine, a health food supplement that stabilises small bowel integrity and stimulates gut repair processes

Background: Zinc carnosine (ZnC) is a health food product claimed to possess health-promoting and gastrointestinal supportive activity. Scientific evidence underlying these claims is, however, limited. Aim: To examine the effect of ZnC on various models of gut injury and repair, and in a clinical trial. Methods: In vitro studies used pro-migratory (wounded monolayer) and proliferation ([3H]-thymidine incorporation) assays of human colonic (HT29), rat intestinal epithelial (RIE) and canine kidney (MDCK) epithelial cells. In vivo studies used a rat model of gastric damage (indomethacin/restraint) and a mouse model of small-intestinal (indomethacin) damage. Healthy volunteers (n = 10) undertook a randomised crossover trial comparing changes in gut permeability (lactulose:rhamnose ratios) before and after 5 days of indomethacin treatment (50 mg three times a day) with ZnC (37.5 mg twice daily) or placebo coadministration. Results: ZnC stimulated migration and proliferation of cells in a dose-dependent manner (maximum effects in both assays at 100 µmol/l using HT29 cells), causing an approximate threefold increase in migration and proliferation (both p<0.01). Oral ZnC decreased gastric (75% reduction at 5 mg/ml) and small-intestinal injury (50% reduction in villus shortening at 40 mg/ml; both p<0.01). In volunteers, indomethacin caused a threefold increase in gut permeability in the control arm; lactulose:rhamnose ratios were (mean (standard error of mean)) 0.35 (0.035) before indomethacin treatment and 0.88 (0.11) after 5 days of indomethacin treatment (p<0.01), whereas no significant increase in permeability was seen when ZnC was coadministered. Conclusion: ZnC, at concentrations likely to be found in the gut lumen, stabilises gut mucosa. Further studies are warranted.

Integration of ERG gene mapping and gene‐expression profiling identifies distinct categories of human prostate cancer

To integrate the mapping of ERG alterations with the collection of expression microarray (EMA) data, as previous EMA analyses have failed to consider the genetic heterogeneity and complex patterns of ERG alteration frequently found in cancerous prostates.

Effect of Ectopic Expression of Rat Trefoil Factor Family 3 (Intestinal Trefoil Factor) in the Jejunum of Transgenic Mice

To further examine the function of the trefoil factor family (TFF), the expression of which is up-regulated at sites of injury, we have produced transgenic mice that chronically express rat TFF3 within the jejunum (using a rat fatty acid-binding protein promoter). The expression of rat TFF3 was limited to the villi of the jejunum and had no effect on base-line morphology. Rat TFF3 expression did result, however, in a reduced sensitivity to indomethacin (85 mg/kg subcutaneously), which only caused a 29% reduction in villus height in transgenics versus 51% reduction in controls (p < 0.01). Indomethacin increased initial intestinal epithelial cell proliferation and migration, but the presence of rat TFF3 caused no additional change in proliferation (bromodeoxyuridine), cell migration ([3H]thymidine and bromodeoxyuridine), apoptosis (terminal deoxyuridine nucleotidyl nick end labeling), or E-cadherin immunostaining. In vitrostudies following changes in resistance of intestinal strips in Ussing chambers (voltage-clamp technique) showed increased base-line resistance in the rat TFF3-expressing region (326 ± 60versus 195 ± 48 ohm·cm2 in controls,p < 0.05) and reduced the fall in resistance following HCl exposure by about 40% (p < 0.01). Overexpression of TFF3 stabilizes the mucosa against noxious agents, supporting its role in mucosal protection/repair. It may therefore provide a novel approach to the prevention and/or treatment of intestinal ulceration.