Benedicte Y. De Winter

Effect of adrenergic and nitrergic blockade on experimental ileus in rats

1 In a rat model of experimental ileus, the effect of blockade of adrenergic and nitrergic neurotransmission was studied on the intestinal transit of Evans blue. 2 Ether anaesthesia and skin incision had no influence on the transit. Laparotomy significantly inhibited the transit of Evans blue. This inhibition was even more pronounced when the small intestine was manipulated. 3 Reserpine (5 mg kg−1), a drug that blocks adrenergic neurotransmission, completely reversed the inhibition of the transit induced by laparotomy but only partially reversed that induced by laparotomy with manipulation of the small intestine. 4 Nω‐nitro‐L‐arginine (L‐NOARG, 5 mg kg−1), a nitric oxide synthase inhibitor, completely reversed the reserpine‐resistant inhibition induced by laparotomy with manipulation of the small intestine. The effect of L‐NOARG was prevented by concomitant administration of L‐arginine. L‐Arginine itself slightly, but significantly enhanced the inhibition. S‐methylisothiourea and aminoguanidine, selective inhibitors of the inducible NO synthase, had no effect on the transit after the three operations. 5 Treatment of the rats with reserpine plus L‐NOARG had no additional effect on the transit after laparotomy as compared to reserpine alone. However, reserpine plus L‐NNA completely reversed the inhibition of the transit induced by laparotomy with manipulation of the small intestine. 6 These findings support the involvement of adrenergic pathways in the pathogenesis of ileus and suggest that the additional inhibitory effect of mechanical stimulation results from an enhanced release of NO by the constitutive NO synthase.

Therapeutic potential of helminth soluble proteins in TNBS‐induced colitis in mice

Background: The hygiene hypothesis suggests an inverse relationship between the incidence of parasitic infections and chronic inflammatory bowel diseases (IBD). We investigated the therapeutic potential of Schistosoma mansoni and Ancylostoma caninum soluble proteins on experimental colitis in mice. Methods: Colitis was induced by intrarectal administration of 10 mg trinitrobenzene sulfonic acid (TNBS) in 30% ethanol. Six hours after TNBS injection, mice were treated intraperitoneally with helminth proteins. Three days later, colonic inflammation was scored based on 5 inflammatory parameters: clinical disease activity, macroscopic and microscopic inflammation score, extent of inflammation, and myeloperoxidase (MPO) activity. To determine immunological pathways induced by S. mansoni proteins we measured cytokine profiles of T‐lymphocytes from colon, mesenteric lymph nodes (MLN), and spleen by real‐time reverse‐transcriptase polymerase chain reaction (RT‐PCR). Results: Control mice showed no signs of inflammation, whereas all inflammatory parameters were significantly increased in mice with colitis. Treatment of mice with colitis with S. mansoni or A. caninum proteins decreased the macroscopic inflammation score, extent of inflammation, and MPO activity. Immunologically, induction of colitis significantly increased expression of IFN‐γ mRNA in the inflamed colon. Treatment with S. mansoni proteins caused a decrease of proinflammatory cytokines (IFN‐γ, IL‐17) in colon and MLN, whereas the production of regulatory cytokines (IL‐10, TGF‐β) increased significantly in colon tissue. Conclusions: Treatment with proteins of S. mansoni and A. caninum ameliorated TNBS‐induced colitis in mice. S. mansoni proteins increased mRNA expression of regulatory cytokines while suppressing expression of proinflammatory cytokines. Therefore, we suggest a therapeutic potential for helminth proteins in the treatment of IBD. (Inflamm Bowel Dis 2009)

Help, I’m losing patient-centredness! Experiences of medical students and their teachers

Medical Education 2010: 44: 662–673

Effect of inhibition of inducible nitric oxide synthase and guanylyl cyclase on endotoxin-induced delay in gastric emptying and intestinal transit in mice

Nitric oxide (NO) is postulated to play a role in endotoxin-induced ileus. We investigated the effect of selective blockade of inducible NO synthase (iNOS) and guanylyl cyclase on endotoxin-induced ileus in mice. Thirty minutes before injection of lipopolysaccharides (LPS), mice were pretreated with L-NAME (Nω-nitro-L-arginine methyl ester, non-selective NOS inhibitor), 1400W (N-(3-(aminomethyl)benzyl)acetamide, selective iNOS inhibitor), ODQ (1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one, guanylyl cyclase inhibitor), dimethyl sulfoxide (DMSO, vehicle), or dexamethasone. After 18 h, general well being deteriorated and the mice developed hypothermia and a significant delay in gastric emptying and intestinal transit as measured by Evans blue. 1400W completely reversed the endotoxin-induced delay in gastric emptying, while L-NAME did not have these beneficial effects. On the contrary, even in control mice, L-NAME delayed gastric emptying. Dexamethasone, DMSO, and ODQ mimicked the effect of 1400W on endotoxin-induced delay in gastric emptying. The endotoxin-induced delay in transit was significantly improved only by 1400W. None of the drugs reversed the hypothermia. In LPS mice treated with L-NAME, the behavior scale increased even further, while it decreased after treatment with 1400W. In conclusion, selective inhibition of iNOS reverses the endotoxin-induced delay in gastric emptying and transit and improves general well being. The pathway used by NO, derived from iNOS, may involve inhibition of guanylyl cyclase or radical scavenging.

Intestinal mast cells in gut inflammation and motility disturbances

Mast cells may be regarded as prototypes of innate immune cells that can be controlled by neuronal mediators. Their activation has been implicated in many types of neuro-inflammatory responses, and related disturbances of gut motility, via direct or indirect mechanisms that involve several mechanisms relevant to disease pathogenesis such as changes in epithelial barrier function or activation of adaptive or innate immune responses. Here we review the evidence for the involvement of mast cells in the inflammation of the bowel wall caused by bowel manipulation that leads to motility disturbances such as postoperative gastroparesis and ileus. Also in IBD there is substantial evidence for the involvement of mast cells and a mast cell-mediated neuroimmune interaction showing an increased number and an increased degranulation of mast cells. We discuss the potential of mast cell inhibition as a bona fide drug target to relief postoperative ileus. Further research on mast cell-related therapy either by stabilizing the mast cells or by blocking specific mast cell mediators as adjunctive therapy in IBD is encouraged, bearing in mind that several drugs currently used in the treatment of IBD possess properties affecting mast cell activities. This article is part of a Special Issue entitled: Mast cells in inflammation.

Functional evidence that ATP or a related purine is an inhibitory NANC neurotransmitter in the mouse jejunum: study on the identity of P2X and P2Y purinoceptors involved

Conflicting views exist on whether ATP is a neurotransmitter in the enteric nervous system. We investigated the role of ATP in enteric transmission in circular muscle strips of the mouse jejunum. On PGF2α‐precontracted muscle strips and in the presence of atropine and guanethidine, electrical field stimulation (EFS, 1–8 Hz) of nonadrenergic noncholinergic (NANC) nerves induced transient relaxations that were abolished by the nerve‐conductance blocker tetrodotoxin. The NO synthase blocker L‐nitroarginine (L‐NOARG) partially inhibited the NANC relaxations to EFS, but fast‐twitch relaxations to EFS were still observed in the presence of L‐NOARG. In the presence of L‐NOARG, ATP, the P2X receptor agonist αβMeATP and the P2Y receptor agonist ADPβS relaxed jejunal muscle strips. Tetrodotoxin did not affect the relaxation to ATP and ADPβS, but inhibited that to αβMeATP. The L‐NOARG‐resistant NANC relaxations to EFS were almost abolished by apamin, a blocker of small‐conductance Ca2+ activated K+ channels, and by suramin and PPADS, blockers of P2 purinoceptors. Relaxations to ATP were almost abolished by apamin and suramin but not affected by PPADS. Desensitisation of αβMeATP‐sensitive P2X receptors, the P2X receptor blocker Evans blue and the P2X1,2,3 receptor blocker NF 279 inhibited the L‐NOARG‐resistant NANC relaxations to EFS and that to αβMeATP without affecting the relaxation to ADPβS. Brilliant blue G, a P2X2,5,7 receptor blocker, did not affect the relaxations to EFS. Desensitisation of P2Y receptors and MRS 2179, a P2Y1 receptor blocker, virtually abolished the L‐NOARG‐resistant NANC relaxations to EFS and the relaxation to ADPβS without affecting the relaxation to αβMeATP. Dipyridamole, an adenosine uptake inhibitor, or theophylline and 8‐phenyltheophylline, blockers of P1 and A1 purinoceptors, respectively, did not affect the purinergic NANC relaxations to EFS. Our results suggest that ATP or a related purine acts as an inhibitory NANC neurotransmitter in the mouse jejunum, activating P2 but not P1 purinoceptors. Relaxations to the purinergic NANC neurotransmitter mainly involve P2Y receptors of the P2Y1 subtype that are located postjunctionally. Purinergic NANC neurotransmission also involves P2X receptors, most likely of the P2X1 and P2X3 subtype, located pre‐ and/or postjunctionally.

Alteration of the purinergic modulation of enteric neurotransmission in the mouse ileum during chronic intestinal inflammation

The effect of chronic intestinal inflammation on the purinergic modulation of cholinergic neurotransmission was studied in the mouse ileum. Chronic intestinal inflammation was induced by infection of mice with the parasite Schistosoma mansoni during 16 weeks. S. mansoni infection induced a chronic inflammatory response in the small intestine, which was characterised by intestinal granuloma formation, increased intestinal wall thickness, blunted mucosal villi and an enhanced activity of myeloperoxidase. In control ileum and in chronically inflamed ileum, electrical field stimulation (EFS) of longitudinal muscle strips induced frequency‐dependent contractions that were abolished by tetrodotoxin (TTX) and atropine. Carbachol induced dose‐dependent contractions that were not affected by TTX but abolished by atropine. In control ileum, adenosine and ATP dose‐dependently inhibited the contractions to EFS. Theophylline and 8‐phenyltheophylline, P1 and A1 receptor antagonists respectively, prevented this inhibitory effect of adenosine and ATP. PPADS, DMPX and MRS 1220, antagonists of P2, A2 and A3 receptors, respectively, did not prevent this inhibitory effect of adenosine and ATP. Adenosine and ATP did not affect the contractions to carbachol. The inhibitory effect of adenosine and ATP on contractions to EFS in control ileum was mimicked by the stable adenosine analogue methyladenosine and by the A1‐receptor agonist N(6)‐cyclohexyladenosine, but not by the A3 receptor agonist 2‐Cl IB‐MECA or by the ATP analogues αβ‐methylene‐ATP and ADPβS. The inhibitory effect of adenosine on contractions to EFS was lost after prolonged (90 min) treatment of control ileum with methyladenosine (100 μM). In chronically inflamed ileum, adenosine, methyladenosine, N(6)‐cyclohexyladenosine and ATP all failed to inhibit the cholinergic nerve‐mediated contractions to EFS. Also theophylline, 8‐phenyltheophylline, PPADS, DMPX and MRS 1220 had no effect on the contractions to EFS and carbachol. The loss of effect of adenosine and ATP was still evident after 52 weeks of infection. These results indicate that in physiological conditions neuronal adenosine A1 receptors modulate cholinergic nerve activity in the mouse ileum. However, during chronic intestinal inflammation, this purinergic modulation of cholinergic nerve activity is impaired. This suggests that chronic intestinal inflammation leads to a dysfunction of specific neuronal regulatory mechanisms in the enteric nervous system.

Cannabidiol Reduces Intestinal Inflammation through the Control of Neuroimmune Axis

Enteric glial cells (EGC) actively mediate acute and chronic inflammation in the gut; EGC proliferate and release neurotrophins, growth factors, and pro-inflammatory cytokines which, in turn, may amplify the immune response, representing a very important link between the nervous and immune systems in the intestine. Cannabidiol (CBD) is an interesting compound because of its ability to control reactive gliosis in the CNS, without any unwanted psychotropic effects. Therefore the rationale of our study was to investigate the effect of CBD on intestinal biopsies from patients with ulcerative colitis (UC) and from intestinal segments of mice with LPS-induced intestinal inflammation. CBD markedly counteracted reactive enteric gliosis in LPS-mice trough the massive reduction of astroglial signalling neurotrophin S100B. Histological, biochemical and immunohistochemical data demonstrated that S100B decrease was associated with a considerable decrease in mast cell and macrophages in the intestine of LPS-treated mice after CBD treatment. Moreover the treatment of LPS-mice with CBD reduced TNF-α expression and the presence of cleaved caspase-3. Similar results were obtained in ex vivo cultured human derived colonic biopsies. In biopsies of UC patients, both during active inflammation and in remission stimulated with LPS+INF-γ, an increased glial cell activation and intestinal damage were evidenced. CBD reduced the expression of S100B and iNOS proteins in the human biopsies confirming its well documented effect in septic mice. The activity of CBD is, at least partly, mediated via the selective PPAR-gamma receptor pathway. CBD targets enteric reactive gliosis, counteracts the inflammatory environment induced by LPS in mice and in human colonic cultures derived from UC patients. These actions lead to a reduction of intestinal damage mediated by PPARgamma receptor pathway. Our results therefore indicate that CBD indeed unravels a new therapeutic strategy to treat inflammatory bowel diseases.

Tolerogenic dendritic cell vaccines to treat autoimmune diseases: can the unattainable dream turn into reality?

Autoimmune diseases affect about one in 15 individuals in developed countries and are characterized by a breakdown in immune tolerance. Current therapeutic approaches against destructive immune responses in autoimmune diseases are based on non-specific agents systemically suppressing the function of many immune effector cells. This indiscriminate immunosuppression, however, often causes serious and sometimes life-threatening side effects. Therefore, the need for more specific treatments resulting in lower toxicity and longer-term solutions is high. Because of the established role of dendritic cells (DCs) in maintaining the balance between immunity and tolerance, tolerogenic (tol)DCs might be novel therapeutic targets to prevent undesirable (auto-)immune responses. The idea behind tolDC therapy is that it is a highly targeted, antigen-specific treatment that only affects the auto-reactive inflammatory response. The therapeutic potential of tolDCs has already been proven in experimental animal models of different autoimmune disorders as well as with in vitro experiments using ex vivo generated human tolDCs, thus the challenge remains in bringing tolDC therapy to the clinic, although first clinical trials have been conducted. In this review, we will extensively discuss the use of tolDCs for induction of antigen-specific tolerance in several autoimmune disease settings, from bench to bedside, including currently applied strategies to generate tolDCs as well as technical difficulties and challenges in the field.

Worms and the treatment of inflammatory bowel disease: are molecules the answer?

The lack of exposure to helminth infections, as a result of improved living standards and medical conditions, may have contributed to the increased incidence of IBD in the developed world. Epidemiological, experimental, and clinical data sustain the idea that helminths could provide protection against IBD. Studies investigating the underlying mechanisms by which helminths might induce such protection have revealed the importance of regulatory pathways, for example, regulatory T-cells. Further investigation on how helminths influence both innate and adaptive immune reactions will shed more light on the complex pathways used by helminths to regulate the hosts immune system. Although therapy with living helminths appears to be effective in several immunological diseases, the disadvantages of a treatment based on living parasites are explicit. Therefore, the identification and characterization of helminth-derived immunomodulatory molecules that contribute to the protective effect could lead to new therapeutic approaches in IBD and other immune diseases.