Bioactive recombinant human lactoferrin, derived from rice, stimulates mammalian cell growth.

Today there is a concern about the use of animal source proteins and peptides in cell culture applications due to potential contamination by adventitious infectious pathogens. Recombinant production of these proteins using a plant host provides a safe and cost effective alternative. In this paper, we tested the effect of rice-derived recombinant human lactoferrin (rhLF) on mammalian cell growth. The purified rhLF was partially (about 50%) iron-saturated (pis-rhLF). Chemical modification of pis-rhLF generated apo-rhLF (<10% iron saturation) or holo-rhLF (>90% iron saturation). All three forms of rhLF (pis, apo, holo) promoted growth of intestinal cells (HT-29) measured as [(3)H]-thymidine incorporation or viable cell count, but holo-rhLF was most effective. Holo-rhLF was further tested on hybridoma, osteoblast, and human embryonic kidney cells. Results showed that holo-rhLF promoted cell growth and reduced cell doubling time. The concentration of holo-rhLF in media was critical in promoting cell growth and each cell line had different concentration dependence with the most effective range from 5 to 200 mg/L. The effect of rhLF on antibody production was determined using a hybridoma cell line. Significantly, more antibodies were produced by cells grown with holo-rhLF than cells grown without holo-rhLF. We also compared the effect of holo-rhLF to that of human transferrin, a component commonly used in cell culture media as an iron source. Holo-rhLF was as effective as human transferrin in promoting cell growth and antibody production. Considering all the data obtained, we conclude that rhLF from rice is effective in promoting mammalian cell growth and increasing cell productivity.

Clinical trial: protective effect of a commercial fish protein hydrolysate against indomethacin (NSAID)-induced small intestinal injury.

BACKGROUND: A partially hydrolysed and dried product of pacific whiting fish is marketed as a health food supplement supporting 'intestinal health'. AIM: To examine whether the partially hydrolysed and dried product of pacific whiting fish influenced the small intestinal damaging side effects of the nonsteroidal anti-inflammatory drug, indomethacin. METHODS: Eight human volunteers completed a double-blind, placebo-controlled, crossover protocol of clinically relevant dose of indomethacin (50 mg t.d.s. p.o. for 5 days) with 7 days of fish hydrolysate or placebo starting 2 days prior to indomethacin. Changes in gut permeability were assessed using 5 h urinary lactulose:rhamnose (L/R) ratios. RESULTS: Fish hydrolysate given alone did not affect permeability. In the main study (n = 8), baseline values were similar for both arms (0.28 +/- 0.05 and 0.35 +/- 0.07). Administration of indomethacin (+placebo) caused a fivefold rise in L/R ratios (increasing to 1.54 +/- 0.35), whereas L/R ratios in the same subjects ingesting indomethacin + fish hydrolysate was only 0.59 +/- 0.14 (P < 0.01 vs. indomethacin alone). Dyspeptic symptoms occurred in four of eight subjects taking indomethacin alone, but zero of eight when hydrolysate was co-administered. CONCLUSION: Natural bioactive products (nutriceuticals), such as fish hydrolysates, may provide a novel approach to the prevention and treatment of NSAID-induced and other gastrointestinal injurious conditions.

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 ([(3)H]-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 micromol/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.

Gastroprotective effects of oral nucleotide administration.

BACKGROUND AND AIMS: Nucleotides form the building blocks of DNA and are marketed as dietary supplements, alone or in combination with other ingredients, to promote general health. However, there has been only limited scientific study regarding the true biological activity of orally administered nucleotides. We therefore tested their efficacy in a variety of models of epithelial injury and repair. METHODS: Effects on proliferation ([3H] thymidine incorporation) and restitution (cell migration of wounded monolayers) were analysed using HT29 and IEC6 cells. The ability of a nucleotide mixture to influence gastric injury when administered orally and subcutaneously was analysed using a rat indomethacin (20 mg/kg) restraint model. RESULTS: In both cell lines, cell migration was increased by approximately twofold when added at 1 mg/ml (p<0.01); synergistic responses were seen when a mixture of nucleotides was used. Cell proliferation was stimulated by adenosine monophosphate (AMP) in HT29, but not in IEC6, cells. Gastric injury was reduced by approximately 60% when gavaged at 4-16 mg/ml (p<0.05), concentrations similar to those likely to be found in consumers taking nucleotide supplements. Systemic administration of nucleotides was unhelpful. CONCLUSIONS: Nucleotides possess 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 on their potential benefits (and risks) appear justified.

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 [(3)H] 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 mug/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.

Synergistic effects of systemic trefoil factor family 1 (TFF1) peptide and epidermal growth factor in a rat model of colitis.

Novel therapies for the treatment of colitis are required. We therefore examined the potential value of the trefoil factor family 1 (TFF1) peptide and epidermal growth factor (EGF) alone and in combination. Effects of TFF1- Cys58 +/- EGF on an in vitro HT29 cell wounding model of restitution showed synergistic activity when used in combination. In addition, animals had colitis induced by adding 4% dextran sulphate sodium (DSS) to the drinking water for 7 days and they also received twice daily subcutaneous injections of test peptides. Treatment with TFF1-Cys58 alone (100 microg/kg) reduced histological colitis score by 22%, but the TFF1-Ser58 variant was ineffective. In a second study, TFF1-Cys58 reduced histological colitis score by 15%, EGF (600 microg/kg) by 26%, and an additive response (42% reduction) was demonstrated when used together (P < 0.01 versus either peptide given alone). Similar results were found using tissue myeloperoxidase (MPO) activity as a marker of inflammation. Where clinical risk/benefit seems justified, these initial studies suggest that combination therapy of systemic EGF and TFF peptides may prove useful for treatment of colitis in patients with disease extending beyond the reach of topical (enema) therapy.

Human transforming growth factor alpha (TGF-alpha) is digested to a smaller (1-43), less biologically active, form in acidic gastric juice.

BACKGROUND: Transforming growth factor alpha (TGF-alpha) is a 50 amino acid peptide with potent proliferative and cytoprotective activity present in gastric mucosa and juice. AIMS: To determine the forms and biological activity of natural and recombinant TGF-alpha following incubation with acid pepsin. PATIENTS: Human gastric juice was obtained under basal conditions from patients taking acid suppressants and from volunteers undergoing intragastric neutralisation. METHODS: Samples were analysed using mass spectroscopy and/or high pressure liquid chromatography with radioimmunoassay. Biological activity was determined using thymidine incorporation into rat hepatocytes and an indomethacin/restraint induced gastric damage rat model. RESULTS: TGF-alpha(1-50) is cleaved to TGF-alpha(1-43) by acid pepsin and this is the predominant form in normal gastric juice. However, intragastric neutralisation or taking acid suppressants caused the predominant form to be TGF-alpha(1-50). TGF-alpha(1-43) had only half of the ability to maximally stimulate [(3)H]thymidine incorporation into primary rat hepatocytes (28 177 (1130) DPM/well for 2.16 nM TGF-alpha(1-43) v 63 184 (3536) DPM/well for TGF-alpha(1-50); p<0.001). A similar reduced potency was seen when used in an indomethacin induced rat gastric damage model (0.18 micro mol/kg/h of TGF-alpha(1-43) reduced ulcer area by 19% whereas TGF-alpha(1-50) reduced area by 62%; p<0.001). CONCLUSIONS: TGF-alpha(1-50) is cleaved to the TGF-alpha(1-43) form by acid pepsin, causing 2-5-fold loss of biological activity. Such changes may have relevance to the actions of acid suppressants and the importance of this peptide in both normal and abnormal growth.

Co-administration of the health food supplement, bovine colostrum, reduces the acute non-steroidal anti-inflammatory drug-induced increase in intestinal permeability.

Non-steroidal anti-inflammatory drugs (NSAIDs) are effective analgesics but cause gastrointestinal injury. Present prophylactic measures are suboptimal and novel therapies are required. Bovine colostrum is a cheap, readily available source of growth factors, which reduces gastrointestinal injury in rats and mice. We therefore examined whether spray-dried, defatted colostrum could reduce the rise in gut permeability (a non-invasive marker of intestinal injury) caused by NSAIDs in volunteers and patients taking NSAIDs for clinical reasons. Healthy male volunteers (n=7) participated in a randomized crossover trial comparing changes in gut permeability (lactulose/rhamnose ratios) before and after 5 days of 50 mg of indomethacin three times daily (tds) per oral with colostrum (125 ml, tds) or whey protein (control) co-administration. A second study examined the effect of colostral and control solutions (125 ml, tds for 7 days) on gut permeability in patients (n=15) taking a substantial, regular dose of an NSAID for clinical reasons. For both studies, there was a 2 week washout period between treatment arms. In volunteers, indomethacin caused a 3-fold increase in gut permeability in the control arm (lactulose/rhamnose ratio 0.36+/-0.07 prior to indomethacin and 1.17+/-0.25 on day 5, P<0.01), whereas no significant increase in permeability was seen when colostrum was co-administered. In patients taking long-term NSAID treatment, initial permeability ratios were low (0.13+/-0.02), despite continuing on the drug, and permeability was not influenced by co-administration of test solutions. These studies provide preliminary evidence that bovine colostrum, which is already currently available as an over-the-counter preparation, may provide a novel approach to the prevention of NSAID-induced gastrointestinal damage in humans.

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 ([(3)H]thymidine and bromodeoxyuridine), apoptosis (terminal deoxyuridine nucleotidyl nick end labeling), or E-cadherin immunostaining. In vitro studies 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 +/- 60 versus 195 +/- 48 ohm.cm(2) 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.

Epidermal growth factor reduces multiorgan failure induced by thioacetamide.

BACKGROUND: Multiorgan failure is a severe life threatening state where present therapeutic approaches are suboptimal. Epidermal growth factor (EGF) is a potent stimulant of repair in in vitro and in vivo models. We therefore examined its potential beneficial effect in reducing mortality and injury induced by the noxious agent thioacetamide (TAA). METHODS: Mice (20 per group) were fasted overnight and received a single intraperitoneal dose of human recombinant EGF at 10 or 30 microg/kg or saline (control). Either 30 minutes before or after EGF, all animals also received TAA (40 mg/kg intraperitoneally). Twenty four hours later, surviving animals were killed, tissues collected, and degree of organ injury assessed. RESULTS: Fifty per cent (10/20) of control animals died within the first 24 hour period. Mortality was almost completely prevented by the higher dose of EGF whether given before or after TAA (p<0.01) and was reduced by about 50% with the lower dose of EGF. In control animals, the entire length of the jejunum and ileum had necrosis with or without mucosal denudation. In contrast, necrosis affected only about 10-20% of the total length in EGF treated groups (both p<0.01 v control). Control animals showed marked glomerular tuft collapse, interstitial haemorrhage, and increased plasma creatinine levels. These effects were significantly reduced in animals given EGF (30 microg/kg; p<0.01). All groups showed similar changes in liver histology (centrilobular necrosis) and alanine transaminase levels (10-fold increase). CONCLUSIONS: Although EGF did not prevent the hepatotoxicity associated with TAA, it reduced mortality, renal injury, and gastrointestinal damage. These studies provide preliminary evidence that EGF may be a novel approach for the prevention and/or treatment of multiorgan failure.

Potency and stability of C terminal truncated human epidermal growth factor.

INTRODUCTION: Epidermal growth factor (EGF) is normally present as EGF(1-53). A variety of C terminal truncated forms have been used in preliminary trials for treating gastrointestinal injury but their relative potency and stability when used in a clinical setting are unclear. Therefore, we compared the biological activity of recombinant EGF(1-53), EGF(1-52), EGF(1-51), and the C terminal peptides EGF(44-53) and EGF(49-53). METHODS: Purity of forms was confirmed by mass spectrometry. Bioactivity of the different EGF forms was determined using [methyl-(3)H] thymidine incorporation into primary rat hepatocytes and their ability to reduce indomethacin (20 mg/kg subcutaneously)/restraint induced gastric injury in rats. Stability of EGF peptides was determined by serial sampling from a syringe driver system containing EGF/4% albumin in saline. RESULTS: Biological activity assays of EGF(1-53), EGF(1-52), and EGF(1-51) gave almost identical thymidine uptake dose-response curves (maximal responses increasing baseline uptake from 4400 (600) cpm (mean (SEM)) to about 22 000 (2000) cpm when EGF was added at 1. 6 nM). EGF(44-53) and EGF(49-53) did not stimulate (3)H thymidine uptake. Control rats had 47 (4) mm(2) damage/stomach, EGF(1-51), EGF(1-52), and EGF(1-53) at 0.16 and 0.80 nmol/kg/h each reduced gastric injury by about 50% and 80%, respectively (both doses p<0.01 compared with control but no significant difference between the different forms). EGF was stable at room temperature for seven days but biological activity decreased by 35% and 40% at two and three weeks, respectively (both p<0.01). Exposure to light did not affect bioactivity. CONCLUSION: EGF(1-51) and EGF(1-52) are as biologically active as full length EGF(1-53) but the C terminal penta- and decapeptides are ineffective. Clinical trials of EGF can probably use infusion systems for at least 48 hours at room temperature and with exposure to light, without reducing biological efficacy.

Endogenous peptides and peptide therapy in gut defense and repair.

The gastrointestinal tract shows a remarkable ability to withstand injury. Factors important in maintaining integrity include rapid proliferation, an excellent blood supply and, in the stomach and colon, the presence of a protective mucus layer. When an injury occurs, the healing process consists of an initial phase of cell migration (restitution), followed by an increase in proliferation and remodeling. There is now a large body of evidence suggesting that at least 30 different peptides are involved in maintaining mucosal integrity and in stimulating the repair process. Insights into the pathophysiological function of these peptides can be gained from considering them as belonging to broad groups based on their overall role in maintaining gut growth or in stimulating repair. Considered in this way, they can be usefully compartmentalized into: 1) mucosal integrity peptides, which are predominantly involved in maintaining normal mucosal integrity; 2) luminal surveillance peptides, which are constantly present in the lumen but which only stimulate proliferation and repair at sites of injury; and 3) rapid response peptides, whose production is rapidly regulated at sites of injury and the function of which is to stimulate the repair process. If peptide therapy for gastrointestinal conditions is to be used, potential methods of delivery include oral, systemic and gene therapy; however, orally delivered peptides may be digested in the gut lumen, systemic administration may result in side effects at sites distant to the gut and methodological problems remain when considering gene therapy. Peptides play a key role in maintaining mucosal homeostasis in the gut. Over the next five years, recombinant peptides are likely to be increasingly used to treat a wide variety of gastrointestinal disorders such as inflammatory bowel disease and necrotizing enterocolitis.

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 (Cys(58)-->Ser(58)), 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-(3)H]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.

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 beta 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.

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 micrograms/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 micrograms/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 beta 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.

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.

Effects of keratinocyte growth factor (KGF) on gut growth and repair.

Keratinocyte growth factor (KGF) is a mitogen found throughout the gastrointestinal tract, but its role in gastrointestinal pathophysiology is unclear. The effect of recombinant KGF on gut growth and repair has been examined using a variety of in vivo models. Rats receiving total parenteral nutrition had co-infusions of KGF or control for 6 days. Changes in gut growth (wet weight and vincristine-induced metaphase arrest) were then assessed. The effects of KGF on gastric repair and acid secretion in rats were determined using an indomethacin (20 mg/kg)/restraint model and animals fitted with chronic gastric fistulae. KGF at 0.1, 1, and 3 mg/kg increased gut growth as assessed by wet weight throughout the gastrointestinal tract and increased vincristine-induced accumulation of metaphases in the stomach and small intestine but not in the colon. Plasma gastrin, peptide YY, enteroglucagon, and glucagon-like peptide-1 were all increased, whereas insulin was lowered by KGF (all P < 0.01). KGF was ineffective in reducing indomethacin-induced gastric damage but caused a reduction in basal acid secretion of about 35 and 50 per cent when administered at 0.2 or 5 mg/kg (P < 0.05). These studies support the idea that KGF is involved in the control of proliferation of the gastrointestinal tract. They do not provide evidence, however, for a role in the early reparative process invoked during short-term models of gastrointestinal injury.

Pancreatic spasmolytic polypeptide protects the gastric mucosa but does not inhibit acid secretion or motility.

The objectives of these studies were to examine whether the trefoil peptide porcine pancreatic spasmolytic polypeptide (PSP) had gastric mucosal protectant properties similar to its human equivalent human spasmolytic polypeptide (hSP) and to confirm the antisecretory and antimotility action of the peptide. PSP and recombinant hSP reduced gastric mucosal damage caused by a combination of subcutaneous indomethacin and restraint stress in the conscious rat. At a dose of 500 micrograms/kg bolus plus 500 micrograms.kg-1.h-1 sc, PSP significantly reduced the total area of damage by 58%. PSP at a dose of 150 micrograms/kg iv had no inhibitory effect on pentagastrin-stimulated gastric acid secretion in the perfused stomachs of anesthetized rats. This lack of antisecretory activity was confirmed in vitro using an isolated stomach preparation from the immature rat. PSP and hSP at concentrations up to 800 nM did not inhibit electrically or chemically evoked contractions of the guinea pig ileum and duodenum in vitro. Thus antisecretory and antimotility actions do not underlie the mucosal protectant properties of PSP. PSP did, however, stimulate cell migration, and this may, at least in part, account for its protectant properties.

Distribution and expression of pancreatic secretory trypsin inhibitor and its possible role in epithelial restitution.

Pancreatic secretory trypsin inhibitor (PSTI) is a potent serine protease inhibitor that prevents excessive digestion of the gastrointestinal mucus but may also directly affect epithelial function. We therefore examined the distribution of PSTI in the human adult and fetus using immunohistochemistry and in situ hybridization and examined its effects on cell proliferation and migration in vitro. PSTI peptide and mRNA were found in the exocrine pancreas, mucus-producing cells of the normal gastrointestinal tract, acinar component of the normal breast, and surface epithelial cells at the edge of benign gastric ulcers. Peptide, but not message, was identified in the renal proximal tubule, probably reflecting reabsorption of filtered peptide. Purified human PSTI did not affect proliferation of the human colonic cell line HT-29 but caused a threefold increase in the rate of migration in an in vitro wounding model of restitution. This effect was inhibited by co-administering a PSTI-neutralizing antibody, a transforming growth factor-beta-neutralizing antibody, or an epidermal growth factor receptor-blocking antibody. As PSTI is widely distributed in several human organ systems and stimulates cell migration in vitro, we conclude that PSTI is likely to have additional roles to that of preserving the gastrointestinal mucous layer from excessive digestion.

Transgenic mice that overexpress the human trefoil peptide pS2 have an increased resistance to intestinal damage.

pS2 is a member of the trefoil peptide family, all of which are overexpressed at sites of gastrointestinal injury. We hypothesized that they are important in stimulating mucosal repair. To test this idea, we have produced a transgenic mice strain that expresses human pS2 (hpS2) specifically within the jejunum and examined the effect of this overexpression on proliferation and susceptibility to indomethacin-induced damage. A transgenic mouse was produced by microinjecting fertilized oocytes with a 1.7-kb construct consisting of rat intestinal fatty acid binding protein promoter (positions -1178 to +28) linked to full-length (490 bp) hpS2 cDNA. Screening for positive animals was by Southern blot analysis. Distribution of hpS2 expression was determined by using Northern and Western blot analyses and immunohistochemical staining. Proliferation of the intestinal mucosa was determined by assessing the crypt cell production rate. Differences in susceptibility to intestinal damage were analyzed in animals that had received indomethacin (85 mg/kg s.c.) 0-30 h previously. Expression of hpS2 was limited to the enterocytes of the villi within the jejunum. In the nondamaged intestine, villus height and crypt cell production rate were similar in transgenic and negative (control) litter mates. However, there was a marked difference in the amount of damage caused by indomethacin in control and transgenic animals in the jejunum (30% reduction in villus height in controls vs. 12% reduction in transgenic animals, P < 0.01) but the damage sustained in the non-hpS2-expressing ileal region was similar in control and transgenic animals. These studies support the hypothesis that trefoil peptides are important in stimulating gastrointestinal repair.