Expression of Transient Receptor Potential Ankyrin 1 and Transient Receptor Potential Vanilloid 1 in the Gut of the Peri-Weaning Pig Is Strongly Dependent on Age and Intestinal Site.

Transient receptor potential (TRP) channels contribute to sensory transduction in the body, agonized by a variety of stimuli, such as phytochemicals, and they are predominantly distributed in afferent neurons. Evidence indicates their expression in non-neuronal cells, demonstrating their ability to modulate gastrointestinal function. Targeting TRP channels could potentially be used to regulate gastrointestinal secretion and motility, yet their expression in the pig is unknown. This study investigated TRPA1 and TRPV1 expression in different gut locations of piglets of varying age. Colocalization with enteroendocrine cells was established by immunohistochemistry. Both channels were expressed in the gut mucosa. TRPV1 mRNA abundance increased gradually in the stomach and small intestine with age, most notably in the distal small intestine. In contrast, TRPA1 exhibited sustained expression across ages and locations, with the exception of higher expression in the pylorus at weaning. Immunohistochemistry confirmed the endocrine nature of both channels, showing the highest frequency of colocalization in enteroendocrine cells for TRPA1. Specific co-localization on GLP-1 immunoreactive cells indicated their possible role in GLP-1 release and the concomitant intestinal feedback mechanism. Our results indicate that TRPA1 and TRPV1 could play a role in gut enteroendocrine activity. Moreover, age and location in the gut significantly affected gene expression.

The distribution of interstitial cells of Cajal in the ileum is not altered by infection with Schistosoma mansoni.

BACKGROUND & OBJECTIVES: The interstitial cells of Cajal (ICC) act as pacemakers that generate slow waves and function as a relay between smooth muscle cells of the gastrointestinal (GI) tract. Recent reports indicate the crucial role played by the ICC in defining GI motility during human disease status like pyloric stenosis, Hirschsprung's disease and ulcerative colitis. Experimental data showed that Nippostrongylus infection in the rat caused an altered GI motility pattern accompanied by a complete loss of ICC-deep muscular plexus. The aim of the present study was to delineate if ICC were similarly affected during Schistosoma mansoni infections, thereby responsible for the disturbed GI motility patterns triggered in the afflicted mammalian host. METHODS & RESULTS: Immunohistochemistry was done using whole mounts and sections from naive and S. mansoni infected mice ileum. Primary antibodies detected Kit-immunoreactivity (Kit-ir representing ICC), PGP-9.5 (protein gene product 9.5 representing a neuronal marker), SK3 (ionic channel marker for non-Kit fibroblast like cells), and Cx43 (gap junction protein representing a muscle marker). Single/double immunofluorescence staining and confocal microscopy depicted that muscle thickness (Cx43-ir) and inflammatory infiltrate increased with infection. Kit-ir ICC and SK3-ir fibroblast like cells (FLC) were present at all normal locations as seen in controls and during acute and chronic stages of infection. INTERPRETATION & CONCLUSION: No disappearance of either ICC population was noted. A preferential (although not exclusive) location of inflammatory infiltrate in contact with SK3-ir FLC in the muscle layer was observed. The present study thus delineated that ICC are not affected during S. mansoni infections, and thereby may not be responsible for mediating the disturbed GI motility patterns caused by schistosomiasis.

Prolonged somatostatin therapy may cause down-regulation of SSTR-like GPCRs on Schistosoma mansoni.

BACKGROUND & OBJECTIVES: Chemotherapy with praziquantel remains the only control measure to Schistosoma mansoni infections to date. The neuropeptide hormone somatostatin gives relief from gastrointestinal disturbances, liverpathology, and reduces egg production in S. mansoni infected mice, suggesting an interaction of somatostatin with the parasite rather than with the host alone. Using antibodies directed to epitopes of the seven somatostatin transmembrane receptors (SSTRs), the presence of SSTRs (or proteins that contain these epitopes) was shown on both worm- and egg-stages of S. mansoni. The present study was undertaken to investigate whether SSTRs on S. mansoni displayed homo/heterodimerisation properties as well as agonist induced down-regulation. RESULTS: Somatostatin therapy was effective after two days of treatment with no further reduction in pathology after five days of therapy. Immunohistochemistry performed on parasite sections showed reactivity of the anti-SSTR antibodies to the tegument and internal parts of adult S. mansoni worms. SDS-PAGE-Western blotting identified protein bands of 70-100 and 200-250 kDa molecular weight. Upon carboxymethylation of the sulfhydryl groups of proteins in the worm lysate, a reduction in density of the protein band at 200-250 kDa and an increase in density of the protein band at 70-100 kDa were noted. This suggested that a substantial amount of the proteins detected on the blot are present as a homo/heterodimer. A protein microarray was used to investigate whether somatostatin therapy induced receptor down- or up-regulation on the adult worm of S. mansoni. Slides spotted with primary anti-SSTR antibody were exposed to lysates of worms collected from infected C3H mice that received none, two days or five days somatostatin treatment, followed by a secondary anti-SSTR antibody coupled to a fluorophore. Comparison of the different samples in terms of parasite dilution till when the fluorescence was detectable, and the fluorescence intensity, proved that the proteins detected in the parasite worm have been down-regulated after five days of somatostatin treatment. CONCLUSION: SSTR-like GPCRs are being expressed by adult S. mansoni worms and extended somatostatin treatment may cause down-regulation of these receptors, thus reducing the therapeutic capacities of this neuropeptide. However, the presence of SSTRs on S. mansoni has not yet been proven on a genetic basis. Cross-reactivity of anti-SSTR antibodies with other G-protein coupled receptors (GPCR) thus cannot yet be excluded.

The therapeutic effect of the neuropeptide hormone somatostatin on Schistosoma mansoni caused liver fibrosis.

BACKGROUND: The neuropeptide somatostatin is one of the major regulatory peptides in the central nervous system and the digestive tract. Our recent work has delineated an association between fibrosis and low levels of endogenous somatostatin plasma levels in Schistosoma mansoni infected subjects. Based on these results this paper explores the therapeutic potential of somatostatin in a mouse model of hepatic fibrosis associated with S. mansoni infections. METHODS: Groups of outbred Swiss mice were infected with 100 S. mansoni cercariae, infection maintained till weeks 10 or 14, and then somatostatin therapy delivered in two regimens -- either a one or a two-day treatment. All animals were sacrificed one week after therapy and controlled for liver, spleen and total body weight. Circulating somatostatin levels in mice plasma were measured at the time of sacrifice by means of a radio-immuno assay. GraphPad Prism was used for statistical calculations. RESULTS: Somatostatin administration showed little toxicity, probably due to its short half-life. Total liver and spleen weights of S. mansoni infected animals increased over time, with no changes observed due to somatostatin therapy. Total body weights were decreased after infection but were not affected by somatostatin therapy. Snap frozen liver sections were stained with haematoxylin-eosin or Masson's trichrome to study parasite count, hepatocyte status, granuloma size and cellularity. After somatostatin treatment mean egg counts per liver section (43.76 +/- 3.56) were significantly reduced as compared to the egg counts in untreated mice after 10 weeks of infection (56.01 +/- 3.34) (P = 0.03). Similar significant reduction in parasite egg counts were also observed after somatostatin treatment at 14 weeks of infection (56.62 +/- 3.02) as compared to untreated animals (69.82 +/- 2.77)(P = 0.006). Fibrosis was assessed from the spectrophotometric determination of tissue hydroxyproline. Infection with S. mansoni caused increased hydroxyproline levels (9.37 +/- 0.63 micromol at wk 10; 9.65 +/- 0.96 micromol at wk 14) as compared to uninfected animals (1.06 +/- 0.10 micromol). This significant increase in collagen content (P = 0.01; 0.007 respectively) marks the fibrosis observed at these time points. Treatment with somatostatin resulted in a significant decrease in hydroxyproline levels both at wk 10 (4.76 +/- 0.58 micromol) and at wk 14 (5.8 +/- 1.13 micromol) (P = 0.01; 0.03 respectively). Endogenous somatostatin levels were increased at wk 10 (297 +/- 37.24 pg/ml) and wk 14 (206 +/- 13.30 pg/ml) of infection as compared to uninfected mice (119 +/- 11.99 pg/ml) (P = 0.01; 0.008 respectively). Circulating somatostatin levels in infected animals were not significantly affected by somatostatin treatment. Hepatocyte status remained unaltered and granulomas were not remarkably changed in size or cellularity. CONCLUSION: Our experiments reveal an antifibrotic effect of somatostatin in schistosomiasis. We have previously shown that the somatostatin receptors SSTR2 and SSTR3 are present on the parasite egg and worms. We therefore hypothesize that somatostatin reduces either the number of parasite eggs or the secretion of fibrosis inducing-mediators. Our data suggest somatostatin may have therapeutic potential in S. mansoni mediated liver pathology.

Effect of somatostatin on gastrointestinal contractility in Schistosoma mansoni infected mice.

Schistosoma mansoni infection induces severe gastrointestinal motility disturbances which are characterised by hyperactivity of intestinal muscle, abdominal pain, diarrhoea, vomiting and nausea. During schistosomiasis, the neuropeptide somatostatin is generated within inflammatory granulomas. However, somatostatin is also an important inhibitory modulator of gastrointestinal motility. In the present study, we have investigated the potential of somatostatin to reduce schistosomiasis-induced hyperactivity of gastrointestinal smooth muscle. Organ bath experiments were performed to study the contractility of isolated smooth muscle strips of intestine from control mice and from mice that were infected with S. mansoni for 2, 4, 8 and 16 weeks. Electrical field stimulation (0.5-8 Hz) of enteric nerves induced frequency-dependent neurogenic contractions of cholinergic origin in all regions of the small intestine. Somatostatin (0.1-1 microM) concentration-dependently inhibited the contractions to enteric nerve stimulation in the small intestine from uninfected control mice and from acutely S. mansoni infected mice (2 and 4 weeks of infection). After 8 weeks of infection with S. mansoni, this inhibitory effect of somatostatin was less pronounced and after 16 weeks of infection it was completely abolished. Histology demonstrated that chronic infection of mice with S. mansoni was associated with significant alterations in the musculature of the small intestine. These alterations may be associated with physiological changes in the responsiveness to somatostatin and suggest that the somatostatin neuroregulatory circuit of enteric neurotransmission in the small intestine is disturbed during chronic schistosomiasis mansoni.