Paul A. Pelckmans

A Positive Response to Infliximab in Crohn Disease: Association with a Higher Systemic Inflammation Before Treatment But Not With -308 TNF Gene Polymorphism

Background: Two-thirds to three-fourths of patients with either refractory luminal or fistulizing Crohn disease respond to infliximab treatment. The ability or inability to respond seems to persist over time. Biological characteristics and/or genetic background can influence the response to treatment. The aim was to assess the value of C-reactive protein and TNF-2 serum levels before treatment as well as the TNF -308 gene polymorphism in the prediction of response to infliximab treatment in Crohn disease. Methods: Two-hundredand-twenty-six Crohn disease patients treated in the setting of an expanded access programme to infliximab in Belgium were studied. There were 136 refractory luminal diseases and 90 refractory fistulizing diseases. Luminal diseases were treated with one single infusion; fistulizing diseases with three infusions at weeks 0,2 and 6. A clinical response to treatment was defined as either a Crohn disease activity index 5 mg/l) than in patients with a normal CRP value (<5mg/l) before treatment (76% versus 46%; P = 0.004; OR: 0.26 (0.11-0.63)). Allelic and genotype frequencies for -308 TNF gene polymorphism were not significantly different between responders and non-responders - with the exception of a slightly higher TNF2 frequency in non-responders in luminal disease (22.1 % versus 11.6%; P = 0.04). However, this was not associated with a significant difference in genotype frequencies. Conclusion: A positive clinical response to infliximab was associated with a higher CRP level before treatment in our population of Crohn disease patients, but there was no relevant association with -308 TNF gene polymorphism. We therefore suggest that CRP level may help to identify better candidates for infliximab treatment.

Inflammatory Bowel Disease A Positive Response to Infliximab in Crohn Disease: Association with a Higher Systemic Inflammation Before Treatment But Not With -308 TNF Gene Polymorphism

Background: Two-thirds to three-fourths of patients with either refractory luminal or fistulizing Crohn disease respond to infliximab treatment. The ability or inability to respond seems to persist over time. Biological characteristics and/or genetic background can influence the response to treatment. The aim was to assess the value of C-reactive protein and TNF- α serum levels before treatment as well as the TNF -308 gene polymorphism in the prediction of response to infliximab treatment in Crohn disease. Methods: Two-hundred-and-twenty-six Crohn disease patients treated in the setting of an expanded access programme to infliximab in Belgium were studied. There were 136 refractory luminal diseases and 90 refractory fistulizing diseases. Luminal diseases were treated with one single infusion; fistulizing diseases with three infusions at weeks 0, 2 and 6. A clinical response to treatment was defined as either a Crohn disease activity index <150 (complete) or a drop of 70 points (partial) at week 4, for luminal disease, and as either complete fistula healing (complete) or a decrease of at least 50% of the number of draining fistulas on two consecutive visits between weeks 0 and 18, for fistulizing disease. CRP and serum TNF- α levels were measured at week 0 before treatment and were compared between responders and non-responders. Patients were genotyped for the-308 TNF gene polymorphism, and allelic as well as genotype frequencies were compared between responders and non-responders. Results: There were 73.2% responders (46.4% complete and 26.8% partial) and 26.8% non-responders. Response rates were similar in luminal and fistulizing diseases. CRP level before treatment was significantly higher in responders than in non-responders (16.8 mg/l (5-160) versus 9.6 mg/l (5-143); P = 0.02). Furthermore, response rate was significantly higher in patients with elevated CRP (>5 mg/l) than in patients with a normal CRP value (<5 mg/l) before treatment (76% versus 46%; P = 0.004; OR: 0.26 (0.11-0.63)). Allelic and genotype frequencies for-308 TNF gene polymorphism were not significantly different between responders and non-responders - with the exception of a slightly higher TNF2 frequency in nonresponders in luminal disease (22.1% versus 11.6%; P = 0.04). However, this was not associated with a significant difference in genotype frequencies. Conclusion: A positive clinical response to infliximab was associated with a higher CRP level before treatment in our population of Crohn disease patients, but there was no relevant association with-308 TNF gene polymorphism. We therefore suggest that CRP level may help to identify better candidates for infliximab treatment.

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)

Evidence for nitric oxide as mediator of non-adrenergic non-cholinergic relaxations induced by ATP and GABA in the canine gut.

1 The effects of haemoglobin, and the nitric oxide (NO) biosynthesis‐inhibitors NG‐monomethyl‐l‐arginine (l‐NMMA), its enantiomer d‐NMMA, and NG‐nitro‐l‐arginine (l‐NNA) were investigated on non‐adrenergic non‐cholinergic (NANC)‐mediated relaxation of circular muscle strips of the canine terminal ileum and ileocolonic junction induced by electrical stimulation, adenosine 5′‐triphosphate (ATP), γ‐aminobutyric acid (GABA) and NO. 2 Tetrodotoxin, l‐NMMA and l‐NNA, but not d‐NMMA, inhibited the relaxations induced by electrical stimulation, ATP and GABA, but not those in response to NO. 3 The inhibitory effect of l‐NMMA and l‐NNA was prevented by l‐arginine, but not by d‐arginine. l‐Arginine did not potentiate any of the NANC relaxations. 4 Haemoglobin reduced the relaxation induced by electrical stimulation, ATP and GABA, and abolished those in response to NO. 5 Our results demonstrate that the ATP‐ and GABA‐induced relaxations resulting from stimulaton of intramural NANC neurones, in addition to those induced by electrical impulses, are mediated by NO or a NO releasing substance and thus provide further evidence in support of the proposal that NO is the final inhibitory NANC neurotransmitter in the canine terminal ileum and ileocolonic junction.

The role of nitric oxide in inhibitory non-adrenergic non-cholinergic neurotransmission in the canine lower oesophageal sphincter.

1 The role of nitric oxide (NO) in non‐adrenergic non‐cholinergic (NANC) neurotransmission was studied on circular muscle strips of the canine lower oesophageal sphincter (LOS). Electrical field stimulation evoked frequency‐dependent relaxations, which were resistant to adrenergic and cholinergic blockade and abolished by tetrodotoxin. 2 Exogenous administration of NO induced concentration‐dependent and tetrodotoxin‐resistant relaxations which mimicked those in response to electrical stimulation. 3 NG‐nitro‐l‐arginine (l‐NNA), a stereospecific inhibitor of NO‐biosynthesis, inhibited the relaxations induced by electrical stimulation but not those by exogenous NO or vasoactive intestinal polypeptide (VIP). 4 The effect of l‐NNA was prevented by l‐arginine, the precursor of the NO biosynthesis but not by its enantiomer d‐arginine. 5 Haemoglobin abolished the NO‐induced responses and reduced those evoked by electrical stimulation. 6 Cumulative administration of VIP induced concentration‐dependent relaxations, which were slow in onset and sustained. A complete relaxation to VIP was not achieved and the relaxations were not affected by l‐NNA. 7 In conclusion, our results provide evidence that NANC relaxations are mediated by NO, suggesting NO or a NO releasing substance as the final inhibitory NANC neurotransmitter in the canine LOS.

Bioassay of nitric oxide released upon stimulation of non‐adrenergic non‐cholinergic nerves in the canine ileocolonic junction

1 The release and the nature of the inhibitory non‐adrenergic non‐cholinergic (NANC) neurotransmitter was studied in the canine ileocolonic junction. A circular muscle strip of the canine ileocolonic junction served as donor tissue in a superfusion bioassay in which rings of rabbit aorta with the endothelium removed served as detector tissue. 2 The ileocolonic junction released a labile factor with vasodilator activity upon stimulation of non‐adrenergic non‐cholinergic (NANC) nerves in response to electrical impulses and the nicotinic receptor agonist 1,1‐dimethyl‐4‐phenylpiperazinium (DMPP). This release was respectively frequency‐ and concentration‐dependent. 3 The release was reduced by the blocker of neuronal conductance, tetrodotoxin, and by the inhibitor of the nitric oxide (NO) biosynthesis NG‐nitro‐l‐arginine. The biological activity was enhanced by superoxide dismutase and eliminated by haemoglobin. Hexamethonium abolished only the release in response to DMPP. 4 Injection of adenosine 5′‐triphosphate (ATP) or vasoactive intestinal polypeptide (VIP) onto the cascade induced relaxations of the rabbit aorta but they were different from those induced by NO or the transferable factor. 5 Based on organ bath experiments in which the reactivity of different parts of the circular smooth muscle layer of the ileocolonic junction was investigated, a muscle strip of superficial circular muscle with submucosa was chosen as the detector strip in the bioassay cascade. 6 The ileocolonic junction dose‐dependently relaxed in response to nitroglycerin and NO. NO was much more potent in the rabbit aorta than in the canine ileocolonic junction. Equivalent amounts of the transferable factor, endothelium‐derived relaxing factor and NO, as assessed by the relaxation of the rabbit aorta, failed to affect the ileocolonic junction. 7 In conclusion, our results demonstrate the release of a transferable vasorelaxant factor in response to NANC nerve stimulation which behaves pharmacologically like NO but not like ATP or VIP. Therefore, we suggest that NO or a NO releasing substance is the inhibitory NANC neurotransmitter in the canine ileocolonic junction.

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.

Value of transrectal ultrasonography in Crohn's disease.

Anorectal lesions are common and can develop silently in patients with Crohns disease. Transrectal ultrasound examinations were performed to study 40 healthy individuals and 40 patients with Crohns disease. A rigid linear endorectal probe was used to examine the rectal wall, the perirectal tissues, and the anal sphincter. In healthy individuals, the rectal wall showed five layers with a total diameter of maximum 4 mm. The anal sphincter was clearly visualized as an echo-poor and sharply delineated structure. No pathological lesions were detected perirectally. In Crohns disease, an enlargement of the rectal wall was seen in 16 patients and heterogeneity of the anal sphincter in 19 patients. This technique detected lesions missed with the routine proctological examinations: four pararectal abscesses, two pararectal fistulas, two para-anal abscesses, and one para-anal fistula. In all examined subjects, the anal sphincter increased in breadth during squeezing and in length during straining. It is concluded that transrectal ultrasonography sharply delineates the rectal wall and the anal sphincter at rest and under dynamic conditions and detects unknown abscesses and fistulas in the pararectal and para-anal tissues in patients with Crohns disease.

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.