Cytokine mRNA quantification in histologically normal canine duodenal mucosa by real-time RT-PCR.

CD4(+) T helper cells are important for the regulation of immune responses in the intestinal mucosa and they exert their effects through the secretion of pro-inflammatory and immunomodulatory cytokines. Human patients with inflammatory bowel diseases (IBD) such as Crohn's disease and ulcerative colitis have alterations in the normal intestinal cytokine profile. These cytokine abnormalities have been shown at both the protein and messenger RNA (mRNA) level. The role that mucosal cytokines play in the pathogenesis of canine IBD has only been investigated using semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) analysis of gut tissue, as cytokine antisera are not available for this species. Real-time RT-PCR has been recognised to be a more accurate and sensitive method of quantifying mRNA transcripts, so in this study TaqMan real-time RT-PCR assays for the quantification of mRNA encoding IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-18, IFN-gamma, TNF-alpha and TGF-beta in canine intestinal mucosa were developed. The amount of these templates was quantified in normal canine duodenal mucosa (n = 8). IL-18, TGF-beta and TNF-alpha were found to be the most abundant transcripts, with IL-10 and IFN-gamma present at levels approximately 10-fold less. IL-2, IL-4, IL-5, IL-6 and IL-12 were the least abundant templates, with some RNA samples having no detectable mRNA copies. The methods developed in this study will form the basis of further work investigating the expression of mRNA encoding cytokines in mucosa from dogs with chronic enteropathies. In addition, these real-time PCR assays can also be used for the quantification of canine cytokine mRNA in other diseases.

Measurement of immunoglobulin concentrations in the feces of healthy dogs.

Selective immunoglobulin A (IgA) deficiency is the most common primary immunodeficiency in humans and may be associated with chronic gastrointestinal disease. This observation has led to the suggestion that the high susceptibility of German shepherd dogs (GSD) to chronic enteropathies is related to a deficiency in mucosal IgA production. Relative deficiencies of IgA has been reported in the serum, saliva, tears, and feces of GSD both with and without alimentary disease; however, the findings of different studies are not consistent. The aim of this study was to confirm whether a relative deficiency of IgA exists in the feces of GSD. Feces were collected from healthy GSD (n = 209), Labrador retrievers (n = 96), beagles (n = 19), and miniature schnauzers (n = 32). Fecal IgA, IgM, and IgG were measured by capture enzyme-linked immunosorbent assays. Fecal IgG concentrations in the four breed groups were not significantly different. IgA concentrations were significantly greater in miniature schnauzers than in GSD (P = 0.0003) and Labradors (P = 0.0004) but not significantly different from those in beagles. IgM concentrations were significantly greater in miniature schnauzers than in GSD (P < 0.0001), Labradors (P < 0.0001), and beagles (P = 0.0098). These findings do not support the hypothesis that GSD have a relative deficiency in fecal IgA. The differences in immunoglobulin concentrations measured from a single defecation, between individuals of the same breed and between breeds, as well as the lack of an internal control molecule, make the determination of a normal reference range for all dogs impossible. Therefore, the usefulness of fecal immunoglobulin quantification for the assessment of intestinal immunoglobulin secretion in dogs is limited.

Inter-digestive and post-prandial antro-pyloro-duodenal motor activity in humans: effect of 5-hydroxytryptamine 1 receptor agonism.

BACKGROUND: Little is known about the effect of 5-hydroxytryptamine 1 (5-HT(1)) receptor agonism on the co-ordinated motor activity of the gastric antrum, pylorus and duodenum under fasting and fed conditions. AIM: To evaluate the effect of sumatriptan, a 5-HT(1) agonist, on fasting and fed antro-pyloro-duodenal motility. METHODS: In study 1, antro-pyloro-duodenal motility was recorded for two phase IIIs of the migrating motor complex and then, following either a subcutaneous injection of sumatriptan 6 mg or saline control, for at least one additional phase III in 11 healthy volunteers (21-36 years). In study 2, the post-prandial motility was recorded for 3 h after either a subcutaneous injection of sumatriptan 6 mg or saline control in 10 healthy volunteers (18-36 years). RESULTS: Sumatriptan prolonged the migrating motor complex cycle (P = 0.009) by increasing the duration of phase II (P = 0.02) but not phases I and III. Post-prandially, sumatriptan reduced the activity index (P = 0.017) by reducing the frequency of co-ordinated motor activity involving the antrum and/or the duodenum (P < 0.05). CONCLUSION: 5-HT(1) receptor agonism increases the periodicity of the migrating motor complex and reduces the occurrence of post-prandial co-ordinated motor activity involving the gastric antrum, pylorus and duodenum.

Gut-focused hypnotherapy normalizes disordered rectal sensitivity in patients with irritable bowel syndrome.

BACKGROUND: We have previously shown that hypnotherapy alters rectal sensitivity in some patients with irritable bowel syndrome. However, this previous study used incremental volume distension of a latex balloon, which might be susceptible to subject response bias and might compromise the assessment of compliance. In addition, the study group was symptomatically rather than physiologically defined. AIM: To assess the effect of hypnotherapy on rectal sensitivity in hypersensitive, hyposensitive and normally sensitive irritable bowel syndrome patients using a distension technique (barostat) that addresses these technical issues. METHODS: Twenty-three irritable bowel syndrome (Rome I) patients (aged 24-72 years) were assessed before and after 12 weeks of hypnotherapy in terms of rectal sensitivity, symptomatology, anxiety and depression. Normal values for sensitivity were established in 17 healthy volunteers (aged 20-55 years). RESULTS: Compared with controls, 10 patients were hypersensitive, seven hyposensitive and six normally sensitive before treatment. Following hypnotherapy, the mean pain sensory threshold increased in the hypersensitive group (P = 0.04) and decreased in the hyposensitive group, although the latter failed to reach statistical significance (P = 0.19). Normal sensory perception was unchanged. Sensory improvement in the hypersensitive patients tended to correlate with a reduction in abdominal pain (r = 0.714, P = 0.07). CONCLUSION: Hypnotherapy improves abnormal sensory perception in irritable bowel syndrome, leaving normal sensation unchanged.

Visceral sensation and emotion: a study using hypnosis.

BACKGROUND AND OBJECTIVES: We have previously shown that hypnosis can be used to study the effect of different emotions on the motility of the gastrointestinal tract. These studies demonstrated that both anger and excitement increased colonic motility while happiness led to a reduction. The purpose of this study was to investigate the effect of hypnotically induced emotion on the visceral sensitivity of the gut. METHODS: Sensory responses to balloon distension of the rectum and compliance were assessed in 20 patients with irritable bowel syndrome (IBS) (aged 17-64 years; 17 female) diagnosed by the Rome I criteria. Patients were studied on four separate occasions in random order either awake (control) or in hypnosis, during which anger, happiness, or relaxation (neutral emotion) were induced. RESULTS: Hypnotic relaxation increased the distension volume required to induce discomfort (p=0.05) while anger reduced this threshold compared with relaxation (p<0.05), happiness (p<0.01), and awake conditions (p<0.001). Happiness did not further alter sensitivity from that observed during relaxation. There were no associated changes in rectal compliance or wall tension. CONCLUSIONS: Further to our previous observations on motility, this study shows that emotion can also affect an IBS patient's perception of rectal distension and demonstrates the critical role of the mind in modulating gastrointestinal physiology. These results emphasise how awareness of the emotional state of the patient is important when either measuring visceral sensitivity or treating IBS.