Leen Thielemans

Alterations in Mucosal Immunity Identified in the Colon of Patients With Irritable Bowel Syndrome

BACKGROUND & AIMS Irritable bowel syndrome (IBS) has been associated with mucosal dysfunction, mild inflammation, and altered colonic bacteria. We used microarray expression profiling of sigmoid colon mucosa to assess whether there are stably expressed sets of genes that suggest there are objective molecular biomarkers associated with IBS. METHODS Gene expression profiling was performed using Human Genome U133 Plus 2.0 (Affymetrix) GeneChips with RNA from sigmoid colon mucosal biopsy specimens from 36 IBS patients and 25 healthy control subjects. Real-time quantitative polymerase chain reaction was used to confirm the data in 12 genes of interest. Statistical methods for microarray data were applied to search for differentially expressed genes, and to assess the stability of molecular signatures in IBS patients. RESULTS Mucosal gene expression profiles were consistent across different sites within the sigmoid colon and were stable on repeat biopsy over approximately 3 months. Differentially expressed genes suggest functional alterations of several components of the host mucosal immune response to microbial pathogens. The most strikingly increased expression involved a yet uncharacterized gene, DKFZP564O0823. Identified specific genes suggest the hypothesis that molecular signatures may enable distinction of a subset of IBS patients from healthy controls. By using 75% of the biopsy specimens as a validation set to develop a gene profile, the test set (25%) was predicted correctly with approximately 70% accuracy. CONCLUSIONS Mucosal gene expression analysis shows there are relatively stable alterations in colonic mucosal immunity in IBS. These molecular alterations provide the basis to test the hypothesis that objective biomarkers may be identified in IBS and enhance understanding of the disease.

The hepatocarcinoma cell line HepG2 does not express a GHS-R1a-type ghrelin receptor.

The interaction of ghrelin, a 28-residue acylated peptide, with the growth hormone secretagogue receptor 1a (GHS-R1a) has been studied mostly in cells expressing a recombinant GHS-R1a. As awareness is growing on the importance to study G protein-coupled receptors in a natural environment, we studied the effect of ghrelin in the human hepatocellular HepG2 cell line because it has been described in literature to respond to ghrelin. Despite extensive efforts, we were not able to confirm mRNA expression of GHS-R1a by reverse transcription PCR, radioligand binding, or ghrelin-induced GHS-R1a receptor activation; therefore, we conclude that HepG2 cells do not express GHS-R1a. On the other hand, we confirmed a modest effect of ghrelin on the up-regulation of IRS-1 phosphorylation, which might suggest the existence of an alternative ghrelin receptor in HepG2 cells.

The effects of NO synthase inhibitors on murine collagen-induced arthritis do not support a role of NO in the protective effect of IFN-γ

DBA/1 mice deficient in expressing the interferon‐γ (IFN‐γ) membrane receptor (IFN‐γR KO mice) are more susceptible to collagen‐induced arthritis (CIA) than wild‐type mice, indicating that endogenous IFN‐γ plays a protective role in the pathogenesis of CIA. In IFN‐γR KO mice, nitric oxide (NO) production during CIA is impaired. Because NO is known to exert immunosuppressive and anti‐inflammatory effects in certain model systems, the protective effect of IFN‐γ might be mediated by NO. Here, we tested in wild‐type mice whether inhibition of NO production by metabolic inhibitors, aminoguanidine (AG) and L‐N‐(1‐iminoethyl)lysine (L‐NIL), could mimic the ablation of the IFN‐γ receptor. A high‐dose regimen of AG supplied in the drinking water inhibited NO production, disease development, and anticollagen antibody production but was also associated with transient body weight loss. At a dose and time regimen that still inhibited NO production but did not cause body weight loss, AG failed to affect disease scores. Treatment with L‐NIL, which more specifically than AG affects inducible NO production, caused a slight increase in anticollagen antibody production although not significantly affecting disease occurrence. These data indicate that the diminished capacity of the IFN‐γR KO mice to produce NO following immunization with collagen is unlikely to account for their higher susceptibility to CIA.

The motilin pharmacophore in CHO cells expressing the human motilin receptor

We performed a structure-activity study with the human motilin receptor, which was recently cloned from thyroid tissue. N-terminal fragments, Ala-analogs of motilin, and motilides were tested in a cell line that expresses the cloned human motilin receptor and apoaequorin. Full potency to induce calcium fluxes was obtained with N-terminal fragments of 14 amino acids. Motilin fragments 1-14 in which residues 1 (Phe), 4 (Ile), and 7 (Tyr) were replaced by Ala showed the largest reduction in potency. Only motilides with an enol configuration had markedly higher potencies compared to erythromycin A. The potencies to induce Ca(2+) fluxes correlated strongly with rabbit binding and contractility data, suggesting that the cloned receptor is indeed the motilin receptor, responsible for contractile effects. Conservation of the motilin pharmacophore in evolution indicates an important physiological role of motilin.

Demonstration of a functional motilin receptor in TE671 cells from human cerebellum

BACKGROUND Our laboratory has described the presence of motilin receptors in the rabbit cerebellum. We discovered its presence in the human TE671 cell line, which is of cerebellar origin. METHODS Cytosolic Ca(2+) fluxes were monitored on a confocal microscope in cells loaded with Indo-1 and stimulated with motilin under various conditions. Binding studies were performed with 125I-[Nle(13)]porcine motilin. Using primers, PCR for the motilin receptor was performed. RESULTS Cells responded to motilin after 45+/-20 s. At different concentrations of motilin (10(-8), 10(-7), 10(-6.5), 10(-6) and 10(-5) M) the percentage of responding cells was 0+/-0, 0.6+/-1.5, 4.9+/-4.7, 21.7+/-15 and 35.7+/-12, respectively. The response was blocked by the motilin antagonists [Phe(3), Nle(13)]po-motilin (0.8+/-1.8%) and GM-109 (0.0+/-0.0%) and mimicked by the agonist ABT-229 (23.6+/-15%). After stimulation with motilin, ABT-229 or [Phe(3),Leu(13)]po-motilin, but not with the antagonist GM-109, cells were desensitized. The response to motilin persisted in Ca(2+)-free solution (22.8+/-14.7%), was not affected by nifedipine (44+/-11%) but was abolished by incubation with thapsigargin (0+/-0%). Neither ryanodine, nor a previous stimulation with caffeine (0+/-0%) in Ca(2+)-free Krebs, nor both could block the response to motilin (28, 32.0+/-5.7, 41.3+/-6.1%, respectively). Binding studies revealed two binding sites for motilin, with a pK(d) of 8.9+/-0.05 and 6.11+/-0.61 (n=4). There were 100 times more low than high affinity receptors per cell. The presence of receptor mRNA was confirmed by PCR. CONCLUSION Functional motilin receptors are present in TE671 cells. The response requires intracellular IP(3)-sensitive Ca(2+) stores. These cells may serve as a model of the central motilin receptor.

Sequence, distribution and quantification of the motilin precursor in the cat.

Due to motilins relation to the migrating motor complex (MMC), the physiology of motilin has been mostly studied in man and dog. The cat does not have an MMC pattern, and little is known about cat motilin. Therefore we identified the cat motilin precursor (GenBank accession no. AF127917) and developed a quantitative polymerase chain reaction (PCR) to explore its distribution in the gastrointestinal tract and in the central nervous system (CNS). The precursor is closely related to the dog precursor and consists of an open reading frame of 348bp encoding the signal peptide (25 amino acids), the motilin sequence (22 amino acids) and the motilin associated peptide (69 amino acids). One amino acid of the signal peptide was subject to gene polymorphism. Quantification of motilin messenger RNA (mRNA) was for the first time achieved. It is most abundant in the gastrointestinal tract, with the highest concentration in the duodenum, the lowest in the colon and is not detectable in the corpus. However an important expression was also observed in several regions of the CNS, except the striatum and cerebral cortex. The highest level was in the hypothalamus (although 23-fold lower than in the duodenum), the lowest level in the pons. Moderate levels were found in the thyroid. These data suggest that the physiological role of motilin may extend beyond its effect on gastrointestinal motility.

Effect of recombinant human interleukin-11 on motilin and substance P release in normal and inflamed rabbits

Recombinant human interleukin-11 (rhIL-11) normalizes depressed smooth muscle tension generation towards motilin and substance P (SP) in rabbits with colitis. The aim of this paper was to evaluate the effect of rhIL-11 treatment on motilin and SP release which could have an effect on the contractility changes. Rabbits received 4, 40, 72 or 720 microg/kg rhIL-11 s.c. or saline, 1 h later a continuous s.c. administration of rhIL-11 was started with or without the induction of colitis (135 mg/kg TNBS) for 5 days. Motilin and SP levels were measured by RIA, motilin mRNA expression by RT-PCR. TNBS-colitis did not affect plasma motilin levels but increased the motilin content of the duodenal mucosa 1.7-fold. rhIL-11 treatment dose-dependently increased plasma motilin levels (720 microg/kg day: 3.5-fold) and the motilin content of the duodenal mucosa (720 microg/kg day: 3.0-fold). The effects of rhIL-11 were similar in normal rabbits and were accompanied by an increased motilin mRNA expression. TNBS-colitis decreased plasma SP levels 2.7-fold and the SP content in the colonic muscle layer 7.1-fold. The decrease in the muscle layer, but not in the plasma, was normalized by rhIL-11 treatment. In normal rabbits, rhIL-11 caused a decrease in plasma SP levels, but had no effect on the tissue content of SP. In conclusion, treatment of inflamed or normal rabbits with rhIL-11 increases plasma and tissue levels of motilin in the duodenal mucosa via an increased expression of motilin in the endocrine cells and induces the release of SP from extrinsic neurons. These changes do not explain the beneficial effect of rhIL-11 on the lowered contractility in inflamed rabbits although a change in balance of neuropeptides may influence gastro-intestinal inflammation.

Motilin and erythromycin-A share a common binding site in the third transmembrane segment of the motilin receptor

UNLABELLED The motilin receptor (MTLR) represents a clinically useful pharmacological target, as agonists binding to the MTLR have gastroprokinetic properties. In order to compare the molecular basis for interaction of the MTLR with motilin and with the non-peptide motilin agonist, erythromycin-A (EM-A), the negatively charged E119 located in the third transmembrane (TM3) region was mutated to D (E119D) and Q (E119Q), respectively, and changes in activity of the mutant receptors were verified. METHODS Each mutant receptor was stably transfected in CHO-cells containing the Ca2+ indicator apo-aequorin. Receptor activation in response to motilin, EM-A and their analogues was assessed by Ca2+-luminescense. RESULTS In the E119Q mutant, the Ca2+ response to motilin and EM-A was abolished while in the E119D mutant it was reduced with 62% (motilin) and 81% (EM-A). The pEC50 values were shifted from 9.65+/-0.03 to 7.41+/-0.09 (motilin) and from 6.63+/-0.12 to 4.60+/-0.07 (EM-A). Acetylation of the N-terminal amine group as in [N-acetyl-Phe]1 mot (1-14), decreased the potency 6.3-fold (WT-MTLR) and 148-fold (E119D). Acetylation of EM-A enol ether induced a more pronounced shift in potency: 7943-fold (WT-MTLR) and 1413-fold (E119D). CONCLUSION The comparable loss of affinity of the mutant receptors for motilin and EM-A indicate that these agonists both interact with the TM3 domain of the MTLR. The results with acetylated derivatives support an ionic interaction between E119 of the MTLR with the N+ of the desosamine sugar in EM-A, but not with the N+ of the free amine group in motilin.