Protease Inhibitors Protect Bovine Colostrum or Chicken Egg Growth Factors from Pancreatic Enzyme Digestion in AGS Cells or Colitic Rats.

BACKGROUND: Bovine colostrum (BC) and chicken egg contain proteins possessing growth factor activity. Epidermal growth factor (EGF) provides much of the pro-reparative activity within BC. Clinical use of orally administered peptide growth factors is hampered by digestion from pancreatic proteases. OBJECTIVES: We examined whether adding a protease inhibitor [soybean trypsin inhibitor (SBTI) or ovomucoid] protected bioactivity of BC ± egg or EGF alone against pancreatic digestion using in vitro and in vivo models. METHODS: BC, egg, or EGF alone or in combination with trypsin inhibitors were tested for proliferative (Alamar blue) activity using human gastric adenocarcinoma (AGS) cells, prior to and after incubation with HCl/pepsin and trypsin/chymotrypsin. Data were analyzed using 2-factor ANOVA. Eight groups (n = 10) of adult female Sprague-Dawley rats (mean: 188.3 ± 0.8 g) received 20 mg/kg/d of BC + egg, 100 µg/d of EGF, 5 mg/d ovomucoid, or 10.8 mg/d SBTI, alone or in combination (in 1 mL 3% NaHCO3) by gavage for 9 d and dextran sodium sulfate (DSS; 5% in drinking water) for the final 7 d. Histology, microscopic damage score, and myeloperoxidase (MPO) were assessed and analyzed using 1-factor ANOVA. RESULTS: Proliferative activities of BC, egg, or EGF were reduced 40-57% by HCl/pepsin exposure and further reduced 14-24% by chymotrypsin/trypsin. Co-addition of SBTI or ovomucoid truncated the decrease in proliferative bioactivity caused by chymotrypsin/trypsin by 54-100% (P < 0.01). In vivo study showed oral EGF alone or protease inhibitors given alone were ineffective in reducing DSS damage, whereas SBTI with EGF or ovomucoid with BC + egg improved protective effects on weight gain, disease activity score, colonic MPO, and histology damage by 3-4-fold (P < 0.01). CONCLUSIONS: Studies using AGS, cells, and Sprague-Dawley rats showed the protease inhibitors ovomucoid and SBTI protected BC, egg, and EGF against loss of bioactivity due to pancreatic enzymes and, when given with NaHCO3, enhanced colonic protection against DSS damage.

Methods to improve efficacy of orally administered bioactive peptides using bovine colostrum as an exemplar.

BACKGROUND: Oral administration of bioactive peptides has potential clinical advantages, but its applicability is limited due to gastric and pancreatic enzyme proteolysis. OBJECTIVE: To examine whether the co-packaging of bovine colostrum (BC), a rich source of IgG, immune and growth factors, with the food additives trehalose (carbohydrate), stearine (fat), casein (protein present in BC) or soy flour (plant based with high protease inhibitory activity) enhances the stability of BC against digestion. DESIGN: Samples alone and in combination (BC+ 10% wt/wt trehalose, stearine, casein or soy) were exposed to HCl/pepsin, followed by trypsin and chymotrypsin ("CT"). Assessment of proliferation used gastric AGS cells (Alamar blue), IgG function measured bovine IgG anti-E.coli binding and ELISAs quantified growth factor constituents. In vivo bioassay assessed ability of BC alone or with soy to reduce injury caused by dextran sodium sulphate (DSS, 4% in drinking water, 7 days, test products started 2 days prior to DSS). RESULTS: Proliferative activity of BC reduced 61% following HCl/pepsin and CT exposure. This was truncated 50% if soy was co-present, and also protected against loss of total IgG, IgG E.coli binding, TGFß, lactoferrin and EGF (all P<0.01 vs BC alone). Co-packaging with trehalose was ineffective in preventing digestion whereas casein or stearine provided some intermediate protective effects. Rats given BC alone showed beneficial effects on weight gain, disease activity index, tissue histology and colonic MPO. Soy alone was ineffective. BC+ soy combination showed the greatest benefit with a dose of 7 mg/kg (6.4 BC + 0.6 soy flour) having the same degree of benefit as using 20 mg/kg BC alone. CONCLUSION: Soy, and to a lesser extent casein, enhanced the biostability of BC against digestive enzymes. Co-packaging of BC with other food products such as soy flour could result in a decreased dose being required, improving cost-effectiveness and patient compliance.

Marked variability in bioactivity between commercially available bovine colostrum for human use; implications for clinical trials.

BACKGROUND: Colostrum, the milk produced during first few days after birth, is rich in immunoglobulins, antimicrobial peptides & growth factors. Multiple clinical trials using bovine colostrum are ongoing but with no assessment of test product bioactivity. OBJECTIVES: To examine variability of bioactivity between 20 commercial colostrum products, contribution of TGFß and EGFR in mediating effects, heat sensitivity of bioactivity and changes in bioactivity of colostrum milkings in the days following calving. DESIGN: In vitro bioactivity used AGS, RIE-1 and Caco-2 cell proliferation (Alamar blue) and migration (wounded monolayers) assays. Changes in colostrum bioactivity determined following addition of TGFß-neutralising antibody, EGFR blocker (Typhostin) and after heating (40-60°C, 60 min). In vivo bioassay assessed ability of colostrum gavage (2ml, 7mg/ml) to reduce gastric damage (NSAID + restraint) in rats. Milkings from 6 cows, days 0-3 post calving were assessed for bioactivity and growth factor concentrations. RESULT: Six-fold differences in pro-proliferative and migratory activity were seen comparing commercial products. Comparison of most- and least-active samples from in vitro studies showed two- to three-fold differences in ability to reduce gastric injury (86% reduction using most-active vs 48% using least-active, p<0.01). Tyrphostin reduced pro-migratory and proliferative activity by 23% and 55%. TGFß neutralisation reduced migratory activity by 83% but did not affect proliferation Heating colostrum powder to 50°C did not affect immunoactivity of haptoglobin, EGF, TGFß, IgG, IGF-1 or betacellulin but decreased bioactivity by >40%. Milking studies showed high bioactivity during first and second milkings on day 0 but 77% reduction by day 3. Changes in total protein, haptoglobin, EGF, TGFß, IgG and IGF-1 paralleled falls in bioactivity. CONCLUSION: Commercial colostrum products possess widely different bioactivity. Variation in heat exposure and/or proportion of day 0 colostrum content may contribute to this. Assessment of colostrum bioactivity has advantages to growth factor quantitation for quality control.

Pasteurized Chicken Egg Powder Stimulates Proliferation and Migration of AGS, RIE1, and Caco-2 Cells and Reduces NSAID-Induced Injury in Mice and Colitis in Rats.

BACKGROUND: Chicken eggs and bovine colostrum contain proteins possessing antimicrobial, immunoregulatory, and growth factor activity. The ability of eggs to influence gut defense and repair is largely unexplored. OBJECTIVE: We examined the effect of pasteurized spray-dried egg on gastrointestinal injury using cell culture and animal models and sought to determine whether adding colostrum provided extra benefit. METHODS: Egg alone, colostrum alone, and a 40:60 egg: colostrum combination were tested for proliferative (Alamar blue) and migratory (wounded monolayer) activity at 1 mg.mL-1 using human colon adenocarcinoma (Caco-2), human gastric cancer (AGS), and rat intestinal epithelioid-1 (RIE1) cells. Four groups of adult male C57BL/6 mice received 20 mg.kg-1.d-1 test products in drinking water for 7 d and indomethacin (85 mg.kg-1, administered subcutaneously) on day 7. Villus height and morphology were assessed. Three groups of adult male Sprague Dawley rats received 20 mg.kg-1.d-1 test product by gavage for 9 d and dextran sodium sulfate (DSS, 4% in drinking water) for the final 7 d. Histology, microscopic damage scoring, and myeloperoxidase were assessed. RESULTS: Egg or colostrum alone caused 3-fold increases in cell proliferation and migration (P < 0.05 compared with baseline). Heating the egg removed its bioactivity. Addition of neutralizing antibodies or tyrphostin showed that ovomucoid, ovalbumin, and the epidermal growth factor receptor mediated the effects of egg (all P < 0.05 compared with egg). Egg reduced shortening of villi caused by indomethacin in mice by 34% and reduced DSS-induced colonic damage in rats by 44-61% (P < 0.05 compared with DSS). Similar results were seen using colostrum alone. In each assay, the 40:60 combination gave improved results compared with the same dose of egg or colostrum alone (P < 0.05). CONCLUSIONS: Studies using AGS, RIE1, and Caco-2 cells, C57BL/6 mice, and Sprague Dawley rats showed protective effects of egg against gut injury. Enhanced results were seen if colostrum and egg were coadministered. Egg powder with or without colostrum may have therapeutic value for prevention and treatment of gut injuries.

Pancreatic secretory trypsin inhibitor reduces multi-organ injury caused by gut ischemia/reperfusion in mice.

Intestinal ischemia/reperfusion (I/R) injury occurs during transplantation, mesenteric arterial occlusion, trauma and shock, causing systemic inflammation, multiple organ dysfunction and high mortality. Pancreatic secretory trypsin inhibitor (PSTI), a serine protease inhibitor expressed in gut mucosa may function as a mucosal protective/repair peptide. We examined whether PSTI affected mesenteric I/R-induced injury. Hypoxia/normoxia (H/N) caused 50% drop in cell viability of AGS, RIE1 and Caco-2 cells but PSTI (10 µg/ml) given prior- or during-hypoxic period improved survival by 50% (p<0.01). Similarly, Caco-2 monolayers exposed to H/N had 300% increase in transepithelial permeability, PSTI truncated this by 50% (p<0.01). Mice underwent mesenteric I/R by clamping jejunum, causing severe mucosal injury, increased apoptotic markers and 3-fold increases in plasma IL-6, IL1ß, TNFα, and tissue lipid peroxidation (MDA) and inflammatory infiltration (MPO) levels. Lungs showed similar significant injury and inflammatory infiltrate markers. Smaller increases in MDA and MPO were seen in kidney & liver. PSTI (20 mg/kg) reduced all injury markers by 50-80% (p<0.01). In vitro and in vivo studies showed PSTI reduced pro-apoptotic Caspase 3, 9 and Baxα levels, normalised Bcl2 and caused additional increases in HIF1α, VEGF and Hsp70 above rises caused by I/R alone (all p<0.01). PSTI also prevented reduction of tight junction molecules ZO1 and Claudin1 (all p<0.01) but did not affect increased ICAM-1 caused by I/R in gut or lung. PSTI may be a useful clinical target to prevent I/R injury.

Oral bovine colostrum supplementation does not increase circulating insulin-like growth factor-1 concentration in healthy adults: results from short- and long-term administration studies.

PURPOSE: Bovine colostrum is available in health food shops and as a sports food supplement and is rich in antibodies and growth factors including IGF-1. World Anti-Doping Agency advises athletes against taking colostrum for fear of causing increased plasma IGF-1. There are also concerns that colostrum may theoretically stimulate malignancy in organs which express IGF-1 receptors. We, therefore, determined changes in plasma IGF-1 levels in subjects taking colostrum or placebo for 1 day, 4 weeks, and 12 weeks. METHODS: Plasma IGF1 levels were determined in healthy males (n = 16) who ingested 40 g bovine colostrum or placebo along with undertaking moderate exercise for total period of 4.5 h. Two further studies followed changes in IGF1 using double-blind, parallel group, placebo-controlled, randomized trials of colostrum or placebo (N = 10 per arm, 20 g/day for 4 weeks and N = 25 colostrum, N = 29 placebo arm 20 g/day for 12 weeks). RESULTS: Baseline IGF1 levels 130 ± 36 ng/ml. 4.5 h protocol showed no effect of colostrum on plasma IGF1 (ANOVA, treatment group: p = 0.400, group × time: p = 0.498, time p = 0.602). Similarly, no effect of colostrum ingestion was seen following 4 week (ANOVA, group: p = 0.584, group × time interaction: p = 0.083, time p = 0.243) or 12 week (ANOVA, group: p = 0.400, group × time interaction: p = 0.498, time p = 0.602) protocol. CONCLUSIONS: Ingestion of standard recommended doses of colostrum does not increase IGF-1 levels in healthy adults, providing additional support for the safety profile of colostrum ingestion.

Zinc carnosine works with bovine colostrum in truncating heavy exercise-induced increase in gut permeability in healthy volunteers.

BACKGROUND: Heavy exercise causes gut symptoms and, in extreme cases, heat stroke that is due to the increased intestinal permeability of luminal toxins. OBJECTIVE: We examined whether zinc carnosine (ZnC), a health-food product taken alone or in combination with bovine colostrum (a natural source of growth factors), would moderate such effects. DESIGN: Eight volunteers completed a 4-arm, double-blind, placebo-controlled crossover protocol (14 d of placebo, ZnC, colostrum, or ZnC plus colostrum) before undertaking standardized exercise 2 and 14 d after the start of treatment. Changes in epithelial resistance, apoptosis signaling molecules, and tight junction (TJ) protein phosphorylation in response to a 2°C rise in body temperature were determined with the use of Caco-2 and HT29 intestinal cells. RESULTS: Body temperature increased 2°C, and gut permeability (5-h urinary lactulose:rhamnose ratios) increased 3-fold after exercise (from 0.32 ± 0.016 baseline to 1.0 ± 0.017 at 14 d; P < 0.01). ZnC or colostrum truncated the rise by 70% after 14 d of treatment. The combination treatment gave an additional benefit, and truncated exercise induced increase at 2 d (30% reduction; P < 0.01). A 2°C temperature rise in in vitro studies caused the doubling of apoptosis and reduced epithelial resistance 3-4-fold. ZnC or colostrum truncated these effects (35-50%) with the greatest response seen with the combination treatment (all P < 0.01). Mechanisms of action included increasing heat shock protein 70 and truncating temperature-induced changes in B cell leukemia/lymphoma-2 associated X protein α and B cell lymphoma 2. ZnC also increased total occludin and reduced phosphorylated tyrosine claudin, phosphorylated tyrosine occludin, and phosphorylated serine occludin, thereby enhancing the TJ formation and stabilization. CONCLUSION: ZnC, taken alone or with colostrum, increased epithelial resistance and the TJ structure and may have value for athletes and in the prevention of heat stroke in military personnel. This trial was registered at www.isrctn.com as ISRCTN51159138.

Pancreatic secretory trypsin inhibitor causes autocrine-mediated migration and invasion in bladder cancer and phosphorylates the EGF receptor, Akt2 and Akt3, and ERK1 and ERK2.

Pancreatic secretory trypsin inhibitor (PSTI) is expressed in most bladder carcinomas, where its pathophysiological relevance is unclear. Using recombinant normal sequence PSTI/tumor-associated trypsin inhibitor (TATI), a variant associated with familial pancreatitis (N34S), an active site-inactivated variant (R18/V19), and immunoneutralization and RNA interference-mediated knockdown techniques, we investigated the actions of PSTI/TATI on cell migration (wounding monolayers), collagen invasion (gel invasion assays), and proliferation (Alamar blue) on 253J, RT4, and HT1376 human bladder carcinoma cell lines. All three forms of PSTI/TATI stimulated migration twofold, and normal sequence PSTI/TATI showed synergistic promigratory effects when added with EGF. Addition of structurally unrelated soybean trypsin inhibitor had no promigratory activity. Similar results were seen using collagen invasion assays, although the active site mutated variant had no proinvasive activity, probably due to reduced Akt2 activation. PSTI/TATI did not stimulate proliferation despite acting, at least partially, through the EGF receptor, as effects of PSTI/TATI were truncated by the addition of an EGF receptor blocking antibody or the tyrosine kinase inhibitor tyrphostin. Cell lines produced endogenous PSTI/TATI, and PSTI/TATI RNA interference knockdown or the addition of PSTI/TATI, EGF receptor, or tyrphostin blocking agents reduced migration and invasion below baseline. PSTI/TATI induced phosphorylation of the EGF receptor, ERK1 and ERK2, Akt2 and Akt3, JNK1, MKK3, and ribosomal protein S6 kinase 1. This profile was more limited than that induced by EGF and did not include Akt1, probably explaining the lack of proproliferative activity. Our findings of autocrine stimulation and synergistic responses between EGF and PSTI/TATI at concentrations found in urine and tissue suggest that PSTI/TATI has pathophysiological relevance.

IL-1ß stimulation of CCD-18co myofibroblasts enhances repair of epithelial monolayers through Wnt-5a.

Subepithelial myofibroblasts are involved in the initiation and coordination of intestinal epithelial repair, but the molecular signaling pathways are largely unknown. The cellular adaptations that occur during repair range from dedifferentiation and migration to proliferation and redifferentiation, in a way that is strongly reminiscent of normal crypt-to-villus epithelial maturation. We therefore hypothesized that Wnt/ß-catenin signaling may have a pivotal role in intestinal epithelial wound repair. We used the established scratch wound method in Caco-2 cells and in nontransformed NCM460 cells to monitor the effects of IL-1ß-stimulated colonic myofibroblasts (CCD-18co) on intestinal epithelial repair, with immunoblotting and immunodepletion to examine the conditioned media. Conditioned media from IL-1ß-stimulated, but not -untreated, myofibroblasts increased Caco-2 wound closure twofold over 24 h. IL-1ß-stimulated myofibroblasts downregulated the differentiation marker sucrase-isomaltase in the Caco-2 cells, whereas the proliferation marker c-myc was upregulated. Array expression profiling identified Wnt-5a as the Wnt-related gene that was most upregulated (28-fold) by IL-1ß stimulation of CCDs. Recombinant Wnt-5a enhanced proliferation of Caco-2 and NCM460 cells. In scratch assays, it increased migration of the leading edge in both cell lines. Wnt-5a immunodepletion of the IL-1ß-CCD conditioned media abrogated the ability to enhance the repair. Wnt-5a often acts through a noncanonical signal transduction pathway. Further experiments supported this pathway in epithelial wound healing: IL-1ß-CCD-mediated repair was not affected by the addition of the canonical Wnt antagonist Dickkopf-1. Furthermore, media from stimulated myofibroblasts (but not Wnt-5a-depleted media) increased c-jun in Caco-2 cell nuclear extracts. Myofibroblast-mediated noncanonical Wnt-5a signaling is therefore important in the dedifferentiation and migration stages of epithelial wound repair.

Dimethyloxalyglycine stimulates the early stages of gastrointestinal repair processes through VEGF-dependent mechanisms.

Dimethyloxalylglycine (DMOG) is an inhibitor of prolyl-4-hydroxylase domain enzymes. Its potential value and mechanism of actions in preventing/treating gastrointestinal injury are, however, poorly understood. We, therefore, examined the effect of DMOG on influencing gut injury and repair using a variety of in vitro and in vivo models. We performed in vitro studies utilising pro-migratory (wounded monolayer) and proliferation (using DNA quantitation) assays of human stomach (AGS) and colonic (HT29) carcinoma cells. Time course studies examined changes in hypoxia-inducible factor (HIF) and vascular endothelial growth factor (VEGF) levels, a growth factor known to be regulated via HIF. In vivo studies utilised a rat gastric (indomethacin, 20 mg/kg and 3 h restraint) damage model. DMOG stimulated migration in a dose-dependent manner, increasing migration twofold when added at 25µM (P<0.01). Additive effects were seen when DMOG was added to cells in hypoxic conditions. DMOG stimulated proliferation dose dependently, increasing proliferation threefold when added at 70 µM (P<0.01). DMOG caused upregulation of both HIF and VEGF within 4 h of administration. Addition of VEGF neutralising antibody truncated migratory and proliferative activity of DMOG by about 70%. Both oral and subcutaneous administration of DMOG decreased gastric injury without influencing intragastric pH (50% reduction in injury when 1 ml gavaged at 0.57 mM, P < 0.01). Indomethacin reduced tissue HIF and VEGF levels but this was prevented if DMOG was present. In conclusion, DMOG stimulates the early phases of gut repair and VEGF-dependent processes appear relevant. Non-peptide factors such as this may be useful to stabilise or repair gut mucosa.

Reparative properties of the traditional Chinese medicine Cordyceps sinensis (Chinese caterpillar mushroom) using HT29 cell culture and rat gastric damage models of injury.

Cordyceps sinensis (CS) is a traditional Chinese medicine and health food used to support many organ systems. It is commercially produced by cultivation in a liquid medium or on a solid (grain/potato) phase. We tested the effects of hot water extracts of liquid-phase and solid-phase commercially grown CS on its ability to influence proliferation (using Alamar blue, an oxidation/reduction indicator), migration (serial-wounded monolayer photomicroscopy), invasion through collagen gel (fluorometric assay) and indomethacin-induced apoptosis (active caspase-3 colorimetric assay) of human colon cancer HT29 cells. An in vivo study used a rat gastric damage model (indomethacin 20 mg/kg and 4 h restraint with oral administration). The CS extract stimulated cell proliferation threefold when added at 10 µg/ml (P < 0·01). Cell migration increased by 69 % and invasion by 17 % when CS was added at 5 mg/ml (P < 0·01). The results also showed that 93 % of the pro-proliferative activity was soluble in ethanol, whereas pro-migratory activity was divided (61:49) into both ethanol-soluble and ethanol-insoluble sub-fractions. Indomethacin-induced apoptosis was not affected by the presence of CS. CS reduced the amount of gastric injury by 63 % when administered orally at 20 mg/ml (P < 0·01), the results being similar to using the potent cytoprotective agent epidermal growth factor at 25 µg/ml (83 % reduction). We conclude that both methods of cultivated CS possess biological activity when analysed using a variety of gut models of injury and repair. Functional foods, such as CS, could provide a novel approach for the prevention and treatment of injury to the bowel.

Pancreatic secretory trypsin inhibitor is a major motogenic and protective factor in human breast milk.

Colostrum is the first milk produced after birth and is rich in immunoglobulins and bioactive molecules. We examined whether human colostrum and milk contained pancreatic secretory trypsin inhibitor (PSTI), a peptide of potential relevance for mucosal defense and, using in vitro and in vivo models, determined whether its presence influenced gut integrity and repair. Human milk was collected from individuals over various times from parturition and PSTI concentrations determined with the use of immunoassay. Human milk samples were analyzed for proliferation and promigratory activity (wounded monolayers) and antiapoptotic activity (caspase-3 activity) with the use of intestinal HT29 cells with or without neutralizing antibodies to PSTI and epidermal growth factor (EGF). Rats were restrained and given indomethacin to induce gastric injury. Effect of gavage with human breast milk with or without neutralizing antibodies on amount of injury were compared with animals receiving a commercial formula feed. PSTI is secreted into human milk, with colostrum containing a much higher concentration of PSTI than human milk obtained later. Human milk stimulated migration and proliferation about threefold and reduced indomethacin-induced apoptosis by about 70-80%. Sixty-five percent of the migratory effect of human milk could be removed by immunoneutralization of PSTI. PSTI worked synergistically with EGF in mediating these effects. Gastric damage in rats was reduced by about 75% in the presence of human milk and was more efficacious than the formula feed (P<0.001). Protective effects of the milk were reduced by about 60% by PSTI immunoneutralization. We concluded that PSTI is secreted into human milk at concentrations that have probable pathophysiological relevance.

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

OBJECTIVE: 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. MATERIALS AND METHODS: We determined genome-wide expression levels with GeneChip Human Exon 1.0 ST arrays (Affymetrix, Santa Clara, CA, USA) using RNA prepared from 35 specimens of prostate cancer from 28 prostates. RESULTS: The expression profiles showed clustering, in unsupervised hierarchical analyses, into two distinct prostate cancer categories, with one group strongly associated with indicators of poor clinical outcome. The two categories are not tightly linked to ERG status. By analysis of the data we identified a subgroup of cancers lacking ERG rearrangements that showed an outlier pattern of SPINK1 mRNA expression. There was a major distinction between ERG rearranged and non-rearranged cancers that involves the levels of expression of genes linked to exposure to beta-oestradiol, and to retinoic acid. CONCLUSIONS: Expression profiling of prostate cancer samples containing single patterns of ERG alterations can provide novel insights into the mechanism of prostate cancer development, and support the view that factors other than ERG status are the major determinants of poor clinical outcome.

Human pancreatic secretory trypsin inhibitor stabilizes intestinal mucosa against noxious agents.

Pancreatic secretory trypsin inhibitor (PSTI) is a serine protease inhibitor, expressed in gut mucosa, whose function is unclear. We, therefore, examined the effects of PSTI on gut stability and repair. Transgenic mice overexpressing human PSTI within the jejunum (FABPi(-1178 to +28) hPSTI construct) showed no change in baseline morphology or morphometry but reduced indomethacin-induced injury in overexpressing hPSTI region by 42% (P < 0.01). Systemic recombinant hPSTI did not affect baseline morphology or morphometry but truncated injurious effects in prevention and recovery rat models of dextran-sodium-sulfate-induced colitis. In vitro studies showed PSTI stimulated cell migration but not proliferation of human colonic carcinoma HT29 or immortalized mouse colonic YAMC cells. PSTI also induced changes in vectorial ion transport (short-circuit current) when added to basolateral but not apical surfaces of polarized monolayers of Colony-29 cells. Restitution and vectorial ion transport effects of PSTI were dependent on the presence of a functioning epidermal growth factor (EGF) receptor because cells with a disrupted (EGFR(-/-) immortalized cells) or neutralized (EGFR blocking antibodies or tyrosine kinase inhibitor) receptor prevented these effects. PSTI also reduced the cytokine release of lipopolysaccharide-stimulated dendritic cells. We conclude that administration of PSTI may provide a novel method of stabilizing intestinal mucosa against noxious agents and stimulating repair after injury.

Effects of mouse and human lipocalin homologues 24p3/lcn2 and neutrophil gelatinase-associated lipocalin on gastrointestinal mucosal integrity and repair.

BACKGROUND & AIMS: The lipocalin superfamily, including the mouse and human homologues 24p3/lcn2 and neutrophil gelatinase-associated lipocalin, show great functional diversity including roles in olfaction, transportation, and prostaglandin synthesis in mammals. Their potential role in maintaining gastrointestinal mucosal integrity and repair is, however, unclear. METHODS: Changes in 24p3/lcn2 expression in the mouse gut in response to various noxious agents were examined using Northern blot, in situ hybridization, and immunohistochemistry. Effects of recombinant 24p3/lcn2 on proliferation ([3H]-thymidine uptake), and restitution (cell-wounding migration) were assessed using human colonic HT29 and HCT116 cells. In addition, the effects of recombinant 24p3/lcn2 on the amount of gastric damage were assessed in rats treated with indomethacin (20 mg/kg) and restraint. RESULTS: Marked up-regulation of expression of 24p3/lcn2 was seen throughout the gut in response to indomethacin or dextran sodium sulfate treatment. Expression was increased particularly in the surface epithelial cells and infiltrating inflammatory cells. Proliferation and restitution assays in the presence of recombinant wild-type sequence neutrophil gelatinase-associated lipocalin, wild-type cys(98)-24p3/lcn2, and mutant ala98-24p3/lcn2 showed that all 3 peptides caused a 3- to 4-fold increase in promigratory activity (P < .01 vs control) but did not influence proliferation. The administration of wild-type cys98-, or mutant ala98-24p3/lcn2 (25 and 50 microg/kg/h, respectively), given via the subcutaneous route, both caused similar reductions in the rat gastric damage model (60% reduction at highest dose, P < .01 vs control), although oral administration was ineffective. CONCLUSIONS: 24p3/lcn2 facilitates mucosal regeneration by promoting cell migration.

Use of growth-hormone-releasing peptide-6 (GHRP-6) for the prevention of multiple organ failure.

Novel therapies for the treatment of MOF (multiple organ failure) are required. In the present study, we examined the effect of synthetic GHRP-6 (growth hormone-releasing peptide-6) on cell migration and proliferation using rat intestinal epithelial (IEC-6) and human colonic cancer (HT29) cells as in vitro models of injury. In addition, we examined its efficacy when given alone and in combination with the potent protective factor EGF (epidermal growth factor) in an in vivo model of MOF (using two hepatic vessel ischaemia/reperfusion protocols; 45 min of ischaemia and 45 min of reperfusion or 90 min of ischaemia and 120 min of reperfusion). In vitro studies showed that GHRP-6 directly influenced gut epithelial function as its addition caused a 3-fold increase in the rate of cell migration of IEC-6 and HT29 cells (P<0.01), but did not increase proliferation ([3H]thymidine incorporation). In vivo studies showed that, compared with baseline values, ischaemia/reperfusion caused marked hepatic and intestinal damage (histological scoring), neutrophilic infiltration (myeloperoxidase assay; 5-fold increase) and lipid peroxidation (malondialdehyde assay; 4-fold increase). Pre-treatment with GHRP-6 (120 microg/kg of body weight, intraperitoneally) alone truncated these effects by 50-85% (all P<0.05) and an additional benefit was seen when GHRP-6 was used in combination with EGF (1 mg/kg of body weight, intraperitoneally). Lung and renal injuries were also reduced by these pre-treatments. In conclusion, administration of GHRP-6, given alone or in combination with EGF to enhance its effects, may provide a novel simple approach for the prevention and treatment of MOF and other injuries of the gastrointestinal tract. In view of these findings, further studies appear justified.