Inflammatie en oxidatieve stress in de pathogenesis van gastro-intestinale dysfunctie

Gastric motility is regulated via central and local neuronal networks of which the latter functions as an autonomic nervous system mediating motility and reflexes without input from the central nervous system. The neuronal plexus mediating autonomic muscle function is called the myenteric plexus. One of the key inhibitory neurotransmitters in this plexus is nitric oxide. Nitric oxide, which is a gaseous neurotransmitter, is released by nitrergic neurons and induces muscle relaxation. There is extensive evidence that loss of nitrergic motor control is a potential consequence of acute gastrointestinal inflammation or diabetes-induced oxidative stress in patients suffering from functional GI disorders and diabetes respectively. In addition, increased small intestinal permeability has been proposed to be the initial trigger of inflammatory alterations or auto-immune diabetes in these patient groups. The eventual loss of nitrergic neurons can be a major factor underlying gastric dysmotility, which may contribute to symptom generation in patients with motor disorders. However, the exact relationship between, increased permeability, persisting (low-grade) inflammation, altered motor control and symptom generation in the stomach remains to be elucidated. The aim of my PhD project was to establish a spontaneous animal model of impaired nitrergic gastric motor control (the BB-rat), to study the mechanisms involved in loss of nitrergic motor function and to evaluate the relevance of our findings into human pathology.The BB-rat consists of a diabetes-resistant (control) and a diabetes-prone (BB-DP) strain, with spontaneous development of diabetes (BB-DPH) after the age of 160 days in about half of the population. Previous data from BB-rats showed ganglionic inflammation, loss of nNOS expression and nitrergic motor control in the small intestine, independently of hyperglycaemia. We now studied neuromuscular function, gastric accommodation and the presence of inflammation in the gastric fundus of BB rats that were 30, 70 and 220 days old. No changes were observed at the age of 30 days when comparing BB-DP with control rats. At the age of 70 days, BB-DP rats displayed a significant loss of muscle relaxation that was attributed to the loss of nitrergic function. Additionally these animals also displayed an increased inflammatory infiltrate at the level of the neuromuscular layer. Similar changes were found in BB-DP and BB-DPH rats of 220 days with an additional loss of nitrergic neurons at this time found. We found gastric accommodation to be impaired in BB-DP animals and this was at least partially attributable to a loss of nitrergic control. In summary, BB-DP rats of 220 days display altered fundic muscle function and impaired accommodation, which is driven by loss of myenteric nitrergic function, and may be driven by a low grade inflammatory insult. These changes in gastric neuromuscular control develop independently from the presence or development of diabetes and without overt muscular lesions. Hence, the non-diabetic BBDP rat provides aspontaneous model for inflammation-induced impaired gastric accommodation.Previous data in the BB-rat showed that there was an additional loss of nitrergic neurons at the level of the small intestine in the hyperglycaemic animals when compared to their normoglycaemic counterparts. Since oxidative stress levels are reported to be increased in diabetic animals and patients, we studied the level of oxidative stress in our model and if this could explain the additional loss of nitrergic neurons in hyperglycaemic animals. We found that oxidative stress levels were increased in hyperglycaemic animals and that an additional loss of nitrergic neurons was present in these animals when compared to their normoglycaemic counterparts. Both normo- and hyperglycaemic animals had an increased inflammation when compared to control animals, suggesting that the hyperglycaemic state causes this additional loss. This was confirmed by the fact that anti-oxidative treatment reduced the additional loss of the nitrergic neurons in these hyperglycaemic animals. These findings suggest that at the level of the small intestine an additional loss of nitrergic neurons could be caused by the increased levels of oxidative stress found in diabetic animals.Increased intestinal permeability has been found prior to the development of diabetes in both animal models and diabetes patients. To date, alterations in permeability have been studied by carbohydrate absorption testing (e.g. lactulose/mannitol), but this does not allow to establish the site of involvement and the underlying molecular changes. Increased duodenal permeability and local immune activation have been demonstrated in functional dyspepsia and can be associated with delayed gastric emptying and dyspeptic symptoms. The aim of this study was to establish whether duodenal permeability is altered in Type-1 and -2 diabetes patients compared to controls and whether this is associated with their symptom burden. We found that duodenal permeability was increased in all diabetes patients and were able to find underlying molecular changes that could explain this. The total amount of mast cells and eosinophils, suggested to contribute to permeability changes in functional GI-patients, was not found to be increased in these patients. Dyspeptic symptoms were increased, but did not significantly correlate with alterations in local permeability. The average gastric emptying rate was not different from healthy controls and although this correlated with the alterations in emptying rate no significance was found. With this study, we are the first to report on local alterations in duodenal permeability in diabetes patients; however these alterations have no or little effect on gastric motility and symptoms.To conclude, we were able to study both inflammation and diabetes induced loss of nitrergic neurons at the level of the myenteric plexus in the GI-tract. This loss induced an impaired gastric function, making this an interesting model to help us understand the pathophysiology that might be present in patients sufferingfrom impaired gastric function. Increased permeability is often suggested to be the initial trigger of immune alterations that eventually lead to an impaired neurogastro function, but most of the data is coming from animal studies and little is known about alterations in patients. We found an increased duodenal permeability in diabetes patients, but this was not associated with presence of inflammatory cells, gastric symptoms or a delay in emptying. This could suggest that there is no direct relationship but more studies with higher n-numbers and specified patient groups should be performed to completely rule out the possible effect of duodenal permeability on emptying rate and symptoms.nrpages: 146status: publishe

CXCR4 and CCR5 ligands cooperate in monocyte and lymphocyte migration and in inhibition of dual-tropic (R5/X4) HIV-1 infection

One of the most important functions of chemokines and their receptors is the regulation of directional migration of leukocytes within tissues. In specific tissue compartments, cells are exposed to multiple chemokines presented in complex dimensional and temporal patterns. Therefore, a leukocyte requires the mechanisms to integrate the various directional signals it receives from different chemoattractants. In this study, we report that CCL3, CCL5, and CCL8, three potent mononuclear cell chemoattractants, are able to synergize with the homeostatic chemokine CXCL12 in the migration of CD14(+) monocytes, CD3(+) T-lymphocytes, or PHA-activated lymphoblasts. In addition, CCL5 augmented the CXCR4 ligand-driven ERK phosphorylation in mononuclear cells. Furthermore, the synergistic effect between CCL5 and CXCL12 in monocyte chemotaxis is inhibited in the presence of specific CCR1 antibody and AMD3100, but not by maraviroc. In HIV-1 infection assays, a combination of CXCL12 and CCL5 cooperated to inhibit the replication of the dual-tropic (R5/X4) HIV-1 HE strain. Finally, although the dual-tropic HIV-1 strain was barely suppressed by AMD3100 or maraviroc alone, HIV-1 infection was completely blocked by the combination of these two receptor antagonists. Our data demonstrate the cooperation between CCL5 and CXCL12, which has implications in migration of monocytes/lymphocytes during inflammation and in HIV-1 infection.status: publishe

The normoglycaemic biobreeding rat: a spontaneous model for impaired gastric accommodation

Impaired gastric accommodation is reported in patients with functional dyspepsia (FD). Previous findings in postinfectious patients with FD suggest that low-grade inflammation and dysfunction of nitrergic nerves play a role in impaired accommodation. To date, spontaneous animal models to study the relationship between these changes are lacking. We hypothesise that the normoglycaemic BioBreeding diabetes-prone (BB-DP) rat provides an animal model of inflammation-induced impaired gastric motor function.status: publishe

Subacute stress and chronic stress interact to decrease intestinal barrier function in rats

Psychological stress increases intestinal permeability, potentially leading to low-grade inflammation and symptoms in functional gastrointestinal disorders. We assessed the effect of subacute, chronic and combined stress on intestinal barrier function and mast cell density. Male Wistar rats were allocated to four experimental groups (n = 8/group): 1/sham; 2/subacute stress (isolation and limited movement for 24 h); 3/chronic crowding stress for 14 days and 4/combined subacute and chronic stress. Jejunum and colon were collected to measure: transepithelial electrical resistance (TEER; a measure of epithelial barrier function); gene expression of tight junction molecules; mast cell density. Plasma corticosterone concentration was increased in all three stress conditions versus sham, with highest concentrations in the combined stress condition. TEER in the jejunum was decreased in all stress conditions, but was significantly lower in the combined stress condition than in the other groups. TEER in the jejunum correlated negatively with corticosterone concentration. Increased expression of claudin 1, 5 and 8, occludin and zonula occludens 1 mRNAs was detected after subacute stress in the jejunum. In contrast, colonic TEER was decreased only after combined stress, and the expression of tight junction molecules was unaltered. Increased mast cell density was observed in the chronic and combined stress condition in the colon only. In conclusion, our data show that chronic stress sensitizes the gastrointestinal tract to the effects of subacute stress on intestinal barrier function; different underlying cellular and molecular alterations are indicated in the small intestine versus the colon.peerreview_statement: The publishing and review policy for this title is described in its Aims & Scope. aims_and_scope_url: http://www.tandfonline.com/action/journalInformation?show=aimsScope&journalCode=ists20status: publishe

A spontaneous animal model of intestinal dysmotility evoked by inflammatory nitrergic dysfunction.

BACKGROUND AND AIMS: Recent reports indicate the presence of low grade inflammation in functional gastrointestinal disorders (FGID), in these cases often called "post-inflammatory" FGIDs. However, suitable animal models to study these disorders are not available. The Biobreeding (BB) rat consists of a diabetes-resistant (BBDR) and a diabetes-prone (BBDP) strain. In the diabetes-prone strain, 40-60% of the animals develop diabetes and concomitant nitrergic dysfunction. Our aim was to investigate the occurrence of intestinal inflammation, nitrergic dysfunction and intestinal dysmotility in non-diabetic animals. METHODS: Jejunal inflammation (MPO assay, Hematoxylin&Eosin staining and inducible nitric oxide synthase (iNOS) mRNA expression), in vitro jejunal motility (video analysis) and myenteric neuronal numbers (immunohistochemistry) were assessed in control, normoglycaemic BBDP and diabetic BBDP rats. To study the impact of iNOS inhibition on these parameters, normoglycaemic BBDP rats were treated with aminoguanidine. RESULTS: Compared to control, significant polymorphonuclear (PMN) cell infiltration, enhanced MPO activity, increased iNOS mRNA expression and a decreased ratio of nNOS to Hu-C/D positive neurons were observed in both normoglycaemic and diabetic BBDP rats. Aminoguanidine treatment decreased PMN infiltration, iNOS mRNA expression and MPO activity. Moreover, it restored the ratio of nNOS to Hu-C/D positive nerves in the myenteric plexus and decreased the abnormal jejunal elongation and dilation observed in normoglycaemic BBDP rats. CONCLUSIONS: Aminoguanidine treatment counteracts the inflammation-induced nitrergic dysfunction and prevents dysmotility, both of which are independent of hyperglycaemia in BB rats. Nitrergic dysfunction may contribute to the pathophysiology of "low-grade inflammatory" FGIDs. Normoglycaemic BBDP rats may be considered a suitable animal model to study the pathogenesis of FGIDs