Thesis (doctoral)--Lund University, 1997. / Added t.p. with thesis statement inserted.

Thesis (doctoral)--Lund University, 1997. / Added t.p. with thesis statement inserted.

Introduction: The role of short-chain fatty acids (SCFAs) in colonic motility is controversial. Germ free (GF) mice are unable to produce SCFAs and serve as a model to study how their absence affects colonic motility. GF transit is slower than controls and colonization of these mice improves gastrointestinal (GI) transit and serotonin (5-HT) levels. Our aim was to determine the role SCFAs play in improving transit, and whether this is dependent on mucosal 5-HT signaling. Methods: Motility was assessed in GF mice via spatiotemporal mapping with intraluminal perfusion of either PBS or SCFA cocktail. Outflow from the colon was recorded to quantify propulsive contractions. Motility was then assessed in TPH1-KO mice with PBS, butyrate and then propionate. GPR43 and 5-HT staining was performed in control and GF colons. Mice were then given chow diet or high sugar diet (HSD) and motility was recorded. Fecal pellets were taken at baseline and just prior to motility experiments and SCFA levels were measured with mass spectrometry. Results: GF mice exhibit significantly lower proportion of propulsive contractions, lower volume of outflow per contraction and slower velocity of contractions compared to controls. SCFAs changed the motility patterns to that of the controls in all parameters. Butyrate administration significantly increased the proportion of propulsive contractions in controls, yet failed to in TPH1 KO mice. Propionate significantly inhibited propulsive contractions in both mice. HSD-fed mice were not different from chow-fed mice in any parameter. No SCFA was significantly reduced, but the change in butyrate concentration was significantly associated with LDC frequency. Conclusions: Our results reveal significant abnormalities in the propulsive nature of colon motor patterns in GF mice, explaining the decreased transit time in in vivo studies. We show that butyrate, not propionate, activates propulsive motility and that this requires mucosal 5-HT, possibly released by ECs. / Thesis / Master of Science (MSc)

10-15% of women take antidepressants during pregnancy. Selective serotonin reuptake inhibitors (SSRIs) are most commonly used for perinatal depression. Perinatal exposure to SSRIs has been shown to disrupt the development of serotonergic signaling pathways in the central nervous system (CNS); however, the effects on the developing enteric nervous system (ENS) remain relatively unexplored. We hypothesized that early life exposure to SSRIs would influence the structural development of the gastrointestinal (GI) tract. We further hypothesized that these structural changes could lead to clinically relevant functional outcomes, such as modifications in susceptibility to inflammation and altered GI motility. Female Wistar rats were given the SSRI, fluoxetine, or vehicle from 2 weeks prior to mating through gestation until weaning. At postnatal day 1 (P1), postnatal day 21 (P21; weaning) and 6 months of age (P6 months) intestines were harvested to assess for structural changes. At P6M, intestines were collected to assess motility in vitro and subsets of the offspring were treated with dextran sulfate sodium (DSS) to assess susceptibility to colitis. At P1, there was a significant decrease in serotonergic neurons in the female colon. At P21, there was a significant increase in serotonergic neurons of both sexes in the colon. At P6M, there was a significant increase in the frequency and velocity of long-distance contractions in the colon when both sexes were combined and an increase in ZO-1 in male colon. In conclusion, SSRI exposure in utero appears to have structural and functional consequences on the developing ENS in the SSRI exposed offspring. The structural consequences are seen in both sexes at P21 and although the structural changes to the ENS resolve by 6 months, motility in the colon continues to be significantly altered. There were no significant differences in chemical colitis, however, we did see difference of quantitative mRNA cytokines, chemokines and extracellular matrix components which may suggest differences in mucosal immune response. The mechanisms by which these changes occur remain to be explored. / Thesis / Master of Science (MSc)

The duodenal mucosa is regularly exposed to acid, digestive enzymes and ingested noxious agents. It is thus critical to maintain a protective barrier to prevent the development of mucosal injury and inflammation, which are often observed in situations when barrier function is impaired. The rate of mucosal bicarbonate secretion, the regulation of epithelial paracellular permeability and motility are each key components of duodenal barrier function. The hormone melatonin is present in high levels in the gastrointestinal tract and it has been hypothesized that melatonin exerts protective properties. This thesis aims to investigate the impact of exogenous melatonin on the regulation of duodenal barrier function and motility in anesthetized rats in vivo. In addition, duodenal tissue was examined histologically and the expression levels of tight junction proteins and melatonin receptors were assessed with qRT-PCR. It was found that melatonin stimulated mucosal bicarbonate secretion and decreased basal paracellular permeability. Exposing the duodenal mucosa to the well-characterized barrier breaker ethanol increased mucosal bicarbonate secretion, paracellular permeability and motility. Omission of luminal Clˉ abolished, while pretreatment with a nicotinic receptor antagonist reduced, the ethanol-induced bicarbonate secretion suggesting that the secretory response to ethanol is meditated via Clˉ/HCO3ˉexchangers and enteric neural pathways. Melatonin reduced the ethanol-induced increases in paracellular permeability and motility either when injected intravenously or when administered in drinking water for two weeks. The actions of melatonin were abolished by the melatonin receptor antagonist luzindole and by nicotinic acetylcholine receptor inhibition. Two weeks oral administration of melatonin up-regulated the expression levels of melatonin receptors, down-regulated the expression of ZO-3 while the expression of ZO-1, ZO-2, claudin 2-4, occludin and myosin light chain kinase were unaffected. Superficial epithelial changes in a few villi were seen in response to ethanol exposure, an effect that was histologically unchanged by melatonin pretreatment. In conclusion, the results suggest that melatonin plays an important role in the neurohumoral regulation of gastrointestinal mucosal barrier function and motility via receptor- and enteric neural-dependent pathways in vivo in rats. Melatonin might be a candidate for treatment of barrier dysfunction in humans.

Diversos trabajos reportan aumento en el número de linfocitos intraepiteliales (LIE), mastocitos y células enterocromafines en pacientes con Sindrome de Intestino Irritable (SII). Muchos de estos hallazgos se basan en el uso de inmunohistoquímica que son de poca disponibilidad en hospitales generales. El objetivo del presente trabajo es estudiar los hallazgos histológicos en la biopsia de colon sólo con histoquimica en pacientes con SII comparándolos con un grupo sin SII. Fueron incluidos 25 pacientes: 16 (64%), con criterios diagnósticos de SII y 9 (36%), sin SII. Se encontró un mayor número de LIE en el grupo de SII (p=0,002). Un grupo de pacientes con criterios Roma III (41,9%) presentó LIE en el rango de Colitis Linfocitica por lo que fueron excluidos de este estudio. No se encontró diferencia estadísticamente significativa en el número de mastocitos, células enterocromafines y eosinofilos. / Several studies have shown increased numbers of intraepithelial lymphocytes (IEL), mast cells, enterochromaffin cells in colonic mucosa of patients with Irritable Bowel Syndrome (IBS). Many of these findings are based is based on immunohistochemistry results, which is not available in general hospitals. Our objective is to study the histological findings observed in colon biopsies from patients with IBS compared with a group without IBS, using only histochemistry. Twenty five (25) patients were included: 16 with IBS and 9 without IBS. We found increased numbers of IEL in patients with IBS (p=0,002). A group of patients with IBS (41.9%) who fulfilled histological criteria for lymphocytic colitis were excluded. There was no significant difference in mast cells, enterochromaffin cells or eosinophils.

Environmental factors such as diet contribute to the pathogenesis of inflammatory bowel disease (IBD). Epidemiological evidence suggests a robust linkage between IBD and the Western diet, which is often characterized by a high intake of food additives. These additives, including synthetic colourants, are widely used, leading to significant human exposure. Allura Red AC (AR) is one of the most popular synthetic colourants, yet little is known about its impact on human health and the role of AR in the pathogenesis of colitis remains elusive. Serotonin (5-hydroxytryptamine; 5-HT), which regulates various gut physiological processes, has been shown to modulate the gut microbiota and enhance susceptibility to colitis. In this thesis, it was discovered that chronic exposure to AR, at a dose found in commonly consumed dietary products, exacerbated dextran sulfate sodium (DSS)-induced colitis and triggered early onset of disease in the CD4+CD45RBhigh T cell-induced colitis model. AR also induced low grade colonic inflammation in naïve C57BL/6 mice. Exposure to AR was associated with increased colonic 5-HT levels and impaired intestinal barrier function via activation of the myosin light chain kinase (MLCK) pathway. However, AR did not promote colitis in mice lacking tryptophan hydroxylase 1 (Tph1), the rate-limiting enzyme responsible for colonic 5-HT synthesis. Further, AR increased colonic 5-HT levels in germ-free (GF) mice and perturbed the gut microbiota composition in specific pathogen-free (SPF) mice. Transfer of this altered microbiota from the dye-exposed SPF mice to GF mice conferred enhanced susceptibility to DSS-induced colitis. Mechanistically, AR induced reactive oxygen species (ROS) generation and promoted 5-HT secretion via the NF-κB pathway in BON cells. Data in this thesis indicate that the widely used synthetic colourant, AR, promotes colitis via colonic 5-HT in microbiota-dependent and -independent pathways. Collectively, these findings provide important information on enhancing public awareness of its detrimental effects on human health. / Thesis / Candidate in Philosophy / Epidemiological and experimental studies suggest a potential link between inflammatory bowel disease (IBD) and diet. The Western diet, often characterized by a high intake of processed foods, is associated with the growing incidence of IBD. Allura Red AC (AR) is a popular artificial food dye found in highly common processed foods, yet little is known about its impact on human health and disease. Serotonin, a key molecule in the gut, has been implicated in large bowel inflammation. Herein, the potential role of AR in the development of colitis was examined. Across multiple models, AR exposure heightened vulnerability to colitis in mice, an effect attenuated by reduced serotonin production in the gut. The effect of AR in enhancing colitis vulnerability occurred via gut microbiota-dependent and -independent pathways. These studies have identified how AR promotes colitis, findings that may advance public health awareness and impact the health of patients with IBD.

<p>At birth the gastrointestinal (GI) tract is rapidly colonized by microbial organisms which exhibit considerable fluctuations in composition across the first two years of life. During this period, the enteric nervous system (ENS) continues to undergo significant structural and functional changes. In the present study, we investigated whether exposure to intestinal microbiota influences the postnatal development of the ENS. We focused our investigations on dopaminergic neurons as they are among the latest populations of neurons to differentiate during enteric development. The myenteric plexus of specific pathogen-free (SPF) and germ-free (GF) mice were examined in whole-mount preparations of the small and large intestine at three time-points: postnatal day 1 (P1), P7, and P28. The density of dopaminergic neurons did not differ significantly between SPF and GF mice in any region of the intestine examined at P1. However, at P7, GF mice had significantly fewer myenteric dopaminergic neurons in the ileum than did SPF mice, and this difference was maintained at P28.</p> <p>The proportion of enteric dopaminergic neurons has been shown to be dependent upon the availability of serotonin. In the GI tract, serotonin can be of neuronal or enterochromaffin (EC) cell origin. We therefore tested the hypothesis that reductions in myenteric dopaminergic neuron densities in the ileum of GF mice were secondary to changes in enteric serotonergic neuron densities or EC cell frequencies. Neither serotonergic neurons nor EC cell numbers were affected by GF status during the postnatal period. The reduction in dopaminergic neurons seen in GF mice must therefore be attributable to a mechanism that has yet to be determined.</p> <p>These findings are consistent with the notion that enteric microbiota can influence the development of late-born neuronal populations. The reduced proportion of dopaminergic neurons in the ileum of GF mice at P7 and P28 may contribute to the previously described altered motility patterns in postnatal GF mice.</p> / Master of Science (MSc)

Mucosal inflammation in conditions ranging from infective acute enteritis or colitis to inflammatory bowel disease (IBD) is accompanied by alteration in enterochromaffin (EC) cell numbers and serotonin (5-hydroxytryptamine; 5-HT) content in the gut. Previously we had shown that CD4+ T cells, via production of T helper (Th)2 cytokines, regulate EC cell biology in the Trichuris muris-infectious colitis model. I further examined the mechanisms of immuno-endocrine interactions in the context of intestinal inflammation. In chapter 3, utilizing human EC cell line and Trichuris muris-mouse model of infectious colitis we identified a critical role of interleukin (IL)-13, a key Th2 cytokine, in increasing EC cell numbers, tryptophan hydroxylase (TPH)1 expression (rate-limiting enzyme of mucosal 5-HT bio-synthesis), and 5-HT production. In chapter 4, we show that IL-13 driven intestinal inflammation is critically dependent on increased 5-HT production using dextran sulfate sodium (DSS) and dinitrobenzene sulphonic acid (DNBS) models of colitis. In DSS-induced colitis, we were the first to identify the increased production of IL-13 and its pathogenic role as IL-13 knockout (IL-13-KO) mice had less severe inflammation compared to wild-type, which was exacerbated following replenishment of 5-HT in IL-13-KO mice. In chapter 5, biopsy examination revealed, higher mucosal IL-13 expression accompanied inflammation in Crohn's disease (CD), which was additionally associated with increased TPH1, 5-HT receptor (5-HTR)3A, 5-HTR7 and decreased 5-HT transporter (5-HTT) expressions. Moreover, CD patients had elevated plasma and platelet-poor plasma 5-HT levels compared to healthy controls (HCs). Furthermore, 5-HTT polymorphism associated genotypes causing inefficiency in 5-HT re-uptake were more common in our patient cohort than HCs. The findings included in this thesis further emphasize the role of immuno-endocrine interactions in intestinal inflammation, which may be a step toward a better diagnosis or management or even a cure for a disease that is of growing concern, and in understanding IBD pathogenesis. / Dissertation / Doctor of Philosophy (PhD) / The gut produces most of the serotonin found in our body, where it regulates many normal functions. A group of special cells, named enterochromaffin cells, produces nearly all of the serotonin in the gut. In diseases of the gut, especially ones that involve inflammation resulting in symptoms like abdominal pain, diarrhea and bleeding, the number of these cells and serotonin concentration are different from that in the normal gut. I found that these changes are controlled by a particular protein produced by immune cells, called interleukin-13, and alteration in serotonin levels, in turn, contributes to the inflammatory process. Our laboratory experiments with cells and animals establish this connection between interleukin-13 and serotonin in gut inflammation. We further confirm this association between interleukin-13 and serotonin in human inflammatory bowel disease. Moreover, we identify a potential genetic cause of these changes in serotonin concentrations which may ultimately result in inflammatory bowel disease.

<p>The duodenal lumen is exposed to aggressive factors with a high potential to cause damage to the mucosa. Bicarbonate secretion by the duodenal mucosa is accepted as the primary important defense mechanism against the hydrochloric acid intermittently expelled from the stomach.</p><p>The present thesis concerns the influence of the central nervous system and the effects of the hormone melatonin on bicarbonate secretion in anesthetized rats in vivo. Effects of melatonin on intracellular calcium signaling by duodenal enterocyte in vitro were examined in tissues of both human and rat origin. The main findings were as follows:</p><p>Melatonin is a potent stimulant of duodenal mucosal bicarbonate secretion and also seems to be involved in the acid-induced stimulation of the secretion. Stimulation elicited in the central nervous system by the α1-adrenoceptor agonist phenylephrine induced release of melatonin from the intestinal mucosa and a four-fold increase in alkaline secretion. The melatonin antagonist luzindole abolished the duodenal secretory response to administered melatonin and to central nervous phenylephrine but did not influence the release of intestinal melatonin. Central nervous stimulation was also abolished by synchronous ligation of the vagal trunks and the sympathetic chains at the sub-laryngeal level. </p><p>Melatonin induced release of calcium from intracellular stores and also influx of extracellular calcium in isolated duodenal enterocytes. Enterocytes in clusters functioned as a syncytium.</p><p>Overnight fasting rapidly and profoundly down-regulated the responses to the duodenal secretagogues orexin-A and bethanechol but not those to melatonin or vasoactive intestinal polypeptide.</p><p>In conclusion, the results strongly suggest that intestinal melatonin plays an important role in central nervous elicited stimulation of duodenal mucosal bicarbonate secretion. Sensitivity of this alkaline secretion to some peripheral stimulators markedly depends on the feeding status.</p>