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.

Dietary microparticles implicated in Crohn's disease can impair macrophage phagocytic activity and act as adjuvants in the presence of bacterial stimuli.

OBJECTIVE AND DESIGN: Western diets regularly expose the gastrointestinal tract (GI) to large quantities ( > 10(12)/day) of man-made, submicron-sized, particles derived from food additives and excipients. These are taken up by M cells, accumulate in gut macrophages, and may influence the aetiology of inflammatory bowel diseases (IBD). MATERIALS: We investigated the effects of common dietary microparticles on the function of macrophages from healthy donors or active Crohn's disease (CD) patients. METHODS: Macrophages were incubated for 24 h with microparticles before being assayed for cytokine production and phagocytic activity. RESULTS: Microparticles alone were non-stimulatory but, in the presence of bacterial antigens such as LPS, they could act as adjuvants to induce potent cytokine responses. Uptake of high concentrations of microparticles also impaired macrophage phagocytic capacity - but not their ability - to take up 2microM fluorescent beads. CONCLUSIONS: While dietary microparticles alone have limited effects on basic macrophage functions, their ability to act as adjuvants could aggravate ongoing inflammatory responses towards bacterial antigens in the GI tract.

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.

Lack of association of MYO9B genetic variants with coeliac disease in a British cohort.

BACKGROUND AND AIMS: Development of coeliac disease involves an interaction between environmental factors (especially dietary wheat, rye, and barley antigens) and genetic factors (there is strong inherited disease susceptibility). The known human leucocyte antigen (HLA)-DQ2 and -DQ8 association explains only a minority of disease heritability. A recent study in the Dutch population suggested that genetic variation in the 3' region of myosin IXB (MYO9B) predisposes to coeliac disease. MYO9B is a Rho family GTPase activating protein involved in epithelial cell cytoskeletal organisation. MYO9B is hypothesised to influence intestinal permeability and hence intestinal antigen presentation. METHODS: Four single nucleotide polymorphisms were chosen to tag all common haplotypes of the MYO9B 3' haplotype block (exons 15-27). We genotyped 375 coeliac disease cases and 1366 controls (371 healthy and 995 population based). All individuals were of White UK Caucasian ethnicity. RESULTS: UK healthy control and population control allele frequencies were similar for all MYO9B variants. Case control analysis showed no significant association of any variant or haplotype with coeliac disease. CONCLUSIONS: Genetic variation in MYO9B does not have a major effect on coeliac disease susceptibility in the UK population. Differences between populations, a weaker effect size than originally described, or possibly a type I error in the Dutch study might explain these findings.

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.

Trial of trefoil factor 3 enemas, in combination with oral 5-aminosalicylic acid, for the treatment of mild-to-moderate left-sided ulcerative colitis.

BACKGROUND: Current treatment of ulcerative colitis is imperfect. Trefoil peptides are known to stimulate repair in many models of injury, including animal models of colitis. AIM: To assess the efficacy of trefoil factor family-3 enema treatment in a clinical trial. METHODS: A total of 16 patients with mild-to-moderate left sided ulcerative colitis were recruited into a double-blind randomized placebo-controlled study. Patients taking steroids or with proctitis only were excluded. Patients received 75 mL enemas containing either human recombinant trefoil factor family-3 (10 mg/mL) or saline alone once a day for 14 days. All patients also received an oral dose-increment of 1.2 g of mesalazine daily above their normal usage. Patients were assessed at 0, 2, 4 and 12 weeks. Remission was defined as Ulcerative Colitis Disease Activity Index of 0 or 1 with no blood in stool. Individual clinical improvement was defined as a Ulcerative Colitis Disease Activity Index reduction of >3. Data was analysed using chi-square test and anova. RESULTS: Median Ulcerative Colitis Disease Activity Index at entry were 8.5 (trefoil factor family-3 group) and 8 (placebo group). Analysed on an intention-to-treat basis, only one patient went into remission (in trefoil factor family-3 group at day 28). Clinical improvement was seen in two trefoil factor family-3 and three placebo patients on day 14 and two patients in each group on day 28. CONCLUSION: Increasing the dose of 5-aminosalicylic acid was moderately effective in reducing the Ulcerative Colitis Disease Activity Index but was insufficient to induce remission. Trefoil factor family-3 enemas were well-tolerated but did not provide additional benefit above that of adding additional 5-aminosalicylic acid alone.

Synergy between TLR9 and NOD2 innate immune responses is lost in genetic Crohn's disease.

BACKGROUND: Nucleotide binding oligomerisation domain 2 (NOD2; also known as CARD15) mutations are associated with Crohn's disease but how mutations cause disease is poorly understood. Innate immune responses are reportedly enhanced by combined NOD2 ligand (muramyl dipeptide, MDP) and Toll-like receptor 4 ligand (TLR4, lipopolysaccharide) stimulation. Intestinal TLR signalling has a dual role-maintaining intestinal homeostasis and protection from injury as well as initiating inflammatory responses. TLR9 is functional in the intestinal epithelium where it is most strongly expressed in Paneth cells. AIMS: To study possible interactions between CpG DNA (TLR9 ligand) and MDP using primary human cells of differing NOD2 genotypes. SUBJECTS: NOD2 wild-type healthy controls (n = 7) and NOD2 homozygous Crohn's disease patients (n = 19), age and sex matched. METHODS: Peripheral blood mononuclear cells were stimulated with CpG DNA and MDP. Cytokines were measured by enzyme linked immunosorbent assay. RESULTS: Tumour necrosis factor alpha (TNF-alpha) and interleukin 8 (IL-8) responses to CpG DNA were similar in NOD2 wild-type and homozygous mutant cells. Concomitant NOD2 stimulation had a marked synergistic effect on CpG DNA induced TNF-alpha responses at 10-100 ng/ml MDP. A mean 2.1-fold increase in CpG DNA induced TNF-alpha responses and a mean 3.7-fold increase in IL-8 responses were observed in NOD2 wild-type cells with 10 ng/ml MDP. This effect was abolished in NOD2 homozygous cells. CONCLUSIONS: NOD2 stimulation normally enhances innate immune responses to CpG DNA. This marked synergistic effect is lost in Crohn's disease patients homozygous for NOD2 mutations, with implications for TLR mediated intestinal homeostasis and inflammation.

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.

Cytokines and growth factor modulators in intestinal inflammation and repair.

Breakdown in gastrointestinal mucosal integrity may be due to increased aggressive factors, including an excessive inflammatory response, decreased mucosal defence or a combination of the two. Our understanding of the control processes underlying these changes has rapidly expanded over the last decade and it is becoming clear that rather than being distinct elements, inflammation and repair are interrelated processes mediated by common cytokines and growth factors, with the division of factors as being a cytokine or a growth factor being somewhat artificial. The use of biological therapies, such as antibodies that cause receptor blockade or administering recombinant growth factors, has now progressed from the laboratory to the clinical arena. This review summarizes current thoughts on the use of these factors in general, but with particular emphasis on inflammatory bowel disease.

Effects of growth factors and receptor blockade on gastrointestinal cancer.

The advent of recombinant peptide technology offers the potential to use one or several peptides to treat a variety of gastrointestinal conditions. However, although cell culture and animal models have shown proof of concept, we are still at a relatively early stage in translating their use to standard clinical practice. Similarly, peptide and non-peptide antagonists of growth factor receptors show great potential as novel antichemotherapy agents. However, their actual place in clinical practice has yet to be established.

Development of a two-site ELISA assay for the dimeric form of human TFF1.

TFF1 is one of three human trefoil proteins expressed principally in the gastrointestinal tract in normal tissues. TFF1 protects the gastric mucosa against damage as a result of its ability to facilitate reconstitution of damaged gastric mucosa and its involvement in the secretion and structure of gastric mucus. The most biologically active molecular form in cell culture and animal models tested is a dimer formed by a disulfide bond between two cysteine residues close to the C terminus of the protein. We have therefore developed an assay for this form of TFF1 which should facilitate its measurement in biological samples.

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.

Effect of epidermal growth factor administration on intestinal cell proliferation, crypt fission and polyp formation in multiple intestinal neoplasia (Min) mice.

Recombinant epidermal growth factor (EGF) may be useful to treat severe ulcerative gastrointestinal injury. There is concern, however, that systemic use of this potent mitogen might increase tumour development and/or progression in susceptible subjects. We therefore examined the effect of chronic administration of systemic EGF to multiple intestinal neoplasia (Min ) mice, who have a genetic defect in the adenomatous polyposis coli (APC) gene, leading to increased polyp development. Min mice (n =26) and wild-type littermates (n =26) received saline or EGF (223 microg of EGF/kg per day) for 4 weeks using subcutaneous osmotic mini-pumps. Cell proliferation and crypt fission were analysed using microdissection techniques and the number and size of polyps in the small and large intestines were determined. EGF increased wet weight and crypt cell proliferation rate by approx. 20% (all P <0.01 compared with the relevant control) in the small intestine and colon of both control and Min mice. In both groups, EGF reduced the colonic fission index by approx. 40% (P <0.01), but did not affect crypt fission in the small intestine. In Min mice, administration of EGF did not increase numbers of polyps or degree of dysplasia, but resulted in a 40% increase in the polyp size in the proximal intestine (P <0.02), but not in the remainder of the small intestine or colon. No polyps were found in control mice given EGF. EGF did not initiate polyp formation in control or Min mice. However, as polyp size is an important determinant for subsequent risk of malignant change in human colon cancer, further studies appear justified.

Current practice in surveillance strategy for patients with Barrett's oesophagus in the UK.

BACKGROUND: Many guidelines exist regarding the surveillance of patients with Barrett's oesophagus. There are limited data, however, with regard to whether practitioners follow these guidelines. METHODS: We assessed current surveillance practice amongst members of the British Society of Gastroenterology using a simple 11-question anonymous survey, mailed to 300 randomly selected members from the British Society of Gastroenterology Handbook. RESULTS: Two hundred and three of the 300 (68%) responded, 76% considering that surveillance was worthwhile. In those who considered surveillance to be worthwhile, 83% used sub-selection based on age, the length of Barrett's oesophagus or the presence of ulcer or stricture. Patients with Barrett's oesophagus of < 3 cm (short-segment) were considered to be inappropriate for surveillance by 62%. Forty-one per cent reported following the 'advised' recommendations of four-quadrant biopsies every 2 cm, whereas 44% followed a 'random and suspicious areas only' protocol. Marked variation was reported in the re-endoscope interval for both low- and high-grade dysplasia. Only 55% reported that two experienced pathologists reviewed all biopsies showing high-grade dysplasia. CONCLUSIONS: Despite the existence of multiple guidelines for Barrett's surveillance, clinical practice varies widely in the UK. This may be due to a lack of knowledge or because gastroenterologists remain unconvinced by the quality of the current evidence of its value. These results have implications for studies attempting to collate data from multiple centres.

Surveillance for Barrett's oesophagus: is there light the end of the metaplastic tunnel?

Metaplastic change of the oesophageal epithelium from normal stratified squamous to columnar-lined with intestinal metaplasia results in an increased risk of development of adenocarcinoma. As a result, endoscopic surveillance has been recommended for the surgically-fit patient. The evidence that these programmes are altering clinical outcome to any major degree, however, is weak. This review highlights some of the areas of controversy and outstanding points that need to be clarified to allow establishment of evidence-based medicine for this condition.