M. Guerci

The endogenous cannabinoid system in the gut of patients with inflammatory bowel disease

Activation of cannabinoid receptors (CBs) by endocannabinoids impacts on a number of gastrointestinal functions. Recent data indicate that CB1 agonists improve 2,4-dinitrobenzene sulfonic acid-induced colitis in mice, thus suggesting a role for the endocannabinoid agonist anandamide (AEA) in protecting the gut against inflammation. We here examined the gut endocannabinoid system in inflammatory bowel disease (IBD) patients, and investigated the ex vivo and in vitro effects of the non-hydrolysable AEA analog methanandamide (MAEA) on the mucosal proinflammatory response. The content of AEA, but not of 2-arachidonoyl-glycerol and N-palmitoylethanolamine, was significantly lower in inflamed than uninflamed IBD mucosa, and this was paralleled by lower activity of the AEA-synthesizing enzyme N-acyl-phosphatidylethanolamine-specific phospholipase D and higher activity of the AEA-degrading enzyme fatty acid amide hydrolase. MAEA significantly downregulated interferon-γ and tumor necrosis factor-α secretion by both organ culture biopsies and lamina propria mononuclear cells. Although these results are promising, further studies are needed to determine the role of cannabinoid pathways in gut inflammation.

Serum hepcidin in inflammatory bowel diseases: biological and clinical significance.

Background: Hepcidin, a peptide produced by hepatocytes, regulates body iron homeostasis. Inflammation increases serum hepcidin, and its determination can be useful in the differential diagnosis of anemias during inflammatory diseases. Methods: We measured serum hepcidin-25 and hepcidin-20 isoforms in 54 patients with inflammatory bowel diseases (IBD) and 54 reference subjects (36 healthy controls and 18 anemic patients without inflammation or renal failure). Disease activity, blood counts, iron status, and erythropoiesis-related parameters were obtained for all study subjects. Results: In IBD hepcidin-25, the peptide bioactive isoform correlated positively with C-reactive protein and serum ferritin; an inverse correlation was observed with transferrin, the soluble transferrin receptor, and the soluble transferrin receptor to Log(ferritin) ratio. Similar correlations were found in reference subjects. Patients with anemia of inflammation had higher hepcidin-25 levels than those with iron deficiency anemia or a combination of iron deficiency anemia and inflammation (P = 0.0061). In patients with inflammation and serum ferritin concentration 100 to 200 ng/mL, hepcidin-25 was low, suggesting that these patients had iron deficiency. A serum hepcidin-25 concentration below 2.0 nM differentiated 85% of patients with iron deficiency anemia (with or without inflammation) from patients with anemia of inflammation. In IBD, hepcidin-20 correlated with both hepcidin-25 and C-reactive protein. Conclusions: In IBD, iron stores, inflammation, and iron requirement for erythropoiesis influence serum hepcidin-25. Hepcidin-25 determination can be useful in the differential diagnosis of IBD-associated anemias. Serum hepcidin-20 is linked to hepcidin-25, but inflammation has an independent regulatory role on its concentration, indicating that hepcidin-20 may have a biological function.

Oxidative stress and thromboxane-dependent platelet activation in inflammatory bowel disease: effects of anti-TNF–α treatment

Patients with inflammatory bowel disease (IBD) are at higher risk of venous thromboembolism and coronary artery disease despite having a lower burden of traditional risk factors. Platelets from IBD patients release more soluble CD40 ligand (CD40L), and this has been implicated in IBD platelet hyper-activation. We here measured the urinary F2-isoprostane 8-iso-prostaglandin (PG)2α (8-iso-PGF2α), urinary 11-dehydro-thromboxane (TX) B2 (11-dehydro-TXB2) and plasma CD40L in IBD patients, and explored the in vitro action of anti-tumour necrosis factor (TNF)-α antibody infliximab on IBD differentiating megakaryocytes. Urinary and blood samples were collected from 124 IBD patients and 37 healthy subjects. Thirteen IBD patients were also evaluated before and after 6-week infliximab treatment. The in vitro effect of infliximab on patient-derived megakaryocytes was evaluated by immunoflorescence microscopy and by flow cytometry. IBD patients had significantly (p<0.0001) higher urinary 8-iso-PGF2α and 11-dehydro-TXB2 as well as plasma CD40L levels than controls, with active IBD patients displaying higher urinary and plasma values when compared to inactive patients in remission. A 6-week treatment with infliximab was associated with a significant reduction of the urinary excretion of 8-iso-PGF2α and 11-dehydro-TXB2 (p=0.008) and plasma CD40L (p=0.001). Infliximab induced significantly rescued pro-platelet formation by megakaryocytes derived from IBD patients but not from healthy controls. Our findings provide evidence for enhanced in vivo TX-dependent platelet activation and lipid peroxidation in IBD patients. Anti-TNF-α therapy with infliximab down-regulates in vivo isoprostane generation and TX biosynthesis in responder IBD patients. Further studies are needed to clarify the implication of infliximab induced-proplatelet formation from IBD megakaryocytes.

Splenic hypofunction in patients with an incidental finding of small-sized spleen at abdominal ultrasound.

Splenic hypofunction is an acquired condition, ranging from a reversible mild hyposplenism to severe splenic atrophy, and may be accompanied by a reduction in spleen size. It is potentially associated with a number of different diseases, including hematological, immunological, gastroenterological and iatrogenic disorders. Functional hyposplenism, as well as asplenia, has been shown to predispose to thromboembolic events, autoimmunity and infectious complications, the latter of which are the most widely recognized risk of these states [1]. In particular, a significantly higher relative risk of pneumococcal sepsis has been demonstrated in patients affected by splenic hypofunction or atrophy [2] due to reduced levels of circulating IgM memory B cells, a unique B cell population that needs the spleen for their survival and generation [3]. Nevertheless, the relationship between splenic function and size in hyposplenic individuals is unknown. Since a small-sized spleen can be easily detected in the course of an abdominal ultrasound examination, we aimed at evaluating splenic function through pitted erythrocyte counting in patients with an incidental finding of atrophic spleen at abdominal ultrasound. Between October 2009 and January 2011, 4,585 patients were referred to the Ultrasound Unit of our Department of Internal Medicine to undergo an abdominal ultrasound evaluation. The most frequent indications were abdominal pain (49 %), malignancy (37 %), liver and biliary disorders (10 %), bowel disorders (7 %), and nephrolithiasis (5 %). All images were obtained by an expert sonographer, using an Esaote model MyLab 70 XVG, with a 1–8 MHz convex probe and an ATL model HDI 3500, a 5.2 MHz convex probe (Philips Medical Systems, Bothel, Washington). The length of the spleen, defined as the maximum distance between the dome and the tip of the spleen, was measured on a longitudinal section with the patient in the supine or right lateral decubitus position and on deep inspiration. Patients, who were diagnosed as having a small spleen: defined as a spleen length B8 cm in men and B7.5 cm in women [4], underwent peripheral blood collection for the evaluation of spleen function. Splenic function was assessed by counting the number of pitted red cells—that is, erythrocytes bearing membrane abnormalities visible under interference phase microscopy as a so-called ‘pit’. Briefly, one drop of fresh venous blood was taken and mixed with 0.5 ml 3 % buffered glutaraldehyde solution, pH 7.4, in a plastic tube. One thousand erythrocytes were examined in a wet preparation (magnification, 1,0009) with a directinterference phase microscope Leica DMLB (Leica Microsystems Wetzlar GmbH, Wetzlar, Germany) equipped with Nomarsky optics by the same experienced observer unaware of the study. The percentage of pitted erythrocytes was calculated and taken as a measure of splenic function (upper limit of normal = 4 %) [5]. A peripheral blood sample was also collected from 52 splenectomized patients, who were used as positive controls. Data were analyzed in the GraphPad Prism statistical PC program (GraphPad Software, San Diego, CA) using the paired t test and the Mann–Whitney U test. Correlations were studied by Spearman’s rank correlation test. A level of p \ 0.05 was considered statistically significant. A small spleen was found in 203 out of 4,585 patients. Only 128 (mean age 61.5 years; 90 females and 38 males) A. Di Sabatino (&) G. Carnevale Maffe L. Brunetti M. Guerci G. R. Corazza First Department of Medicine, Fondazione IRCCS Policlinico San Matteo, University of Pavia, Piazzale Golgi 19, 27100 Pavia, Italy e-mail: a.disabatino@smatteo.pv.it

Association between defective spleen function and primary eosinophilic gastrointestinal disorders

Primary eosinophilic gastrointestinal disorders (EGID) are inflammatory diseases of unknown etiology characterized by eosinophilic mucosal infiltration of one or more segments of the gastrointestinal tract in the absence of known causes of secondary eosinophilia, such as inflammatory, infectious, malignant, or hypersensitivity disorders. The precise incidence of EGID is unknown, due to its rarity, but it is rising in the latest years, probably because of a better recognition and an increasing interest. Although both pathogenic mechanisms and natural history are mostly unrevealed, there seems to be a common association of EGID with atopy, such as IgE-mediated food allergy, asthma, atopic dermatitis, and allergic rhinitis or, to a lesser extent, autoimmunity, such as Hashimoto’s thyroiditis, rheumatoid arthritis, and systemic lupus erythematosus. Recently, we described the first case of primary EGID complicated by spleen hypofunction, an acquired impairment of both the reticuloendothelial and immune functions of the spleen, accompanied or not by a reduction in spleen size, which potentially predisposes to encapsulated bacterial infections, thromboembolism, and autoimmunity. Splenic hypofunction has been described in a number of gastrointestinal disorders, including celiac disease and inflammatory bowel disease, although the mechanisms implicated in its pathogenesis are still unknown. On this basis, we aimed to assess spleen function in a series of consecutively enrolled patients with EGID. Peripheral blood samples were obtained from 21 patients affected by primary EGID at diagnosis (mean age 43.7 years, range 15-79 years). Twenty-two healthy volunteers (mean age 42.5 years, range 26-61 years) and 18 splenectomized patients (mean age 43.1 years, range 24-68 years) were used as negative and positive controls, respectively. Diagnosis of EGID was based on gastrointestinal symptoms associated with a pathologic eosinophilic infiltration of the gastrointestinal wall, without evidence of parasitic infection, other causes of secondary eosinophilic infiltration, and after ruling out hypereosinophilic syndrome. All patients with EGID (3 with esophagitis, 8 with gastroenteritis, and 10 with colitis) underwent upper and/or lower endoscopy with collection of biopsies. Patients with esophagitis were on high-dose proton pump inhibitors at the time of the biopsy. The number of eosinophils in the lamina propria in each high power field (HPF) was counted as the peak of eosinophil count (the highest number of eosinophils per HPF; 40 objective). In most cases of gastroenteritis and colitis, clusters of eosinophils within the lamina propria and occasional intraepithelial eosinophils were also present, and no cases of mucosal erosions or ulcerations were seen. One of 21 patients with EGID has been already described by us. Seven patients had concomitant autoimmune disorder(s), that is, Hashimoto’s thyroiditis (n 1⁄4 5), autoimmune atrophic gastritis (n 1⁄4 2), autoimmune hemolytic anemia (n 1⁄4 1), and rheumatoid arthritis (n 1⁄4 1). To assess the response to therapy, in 10 patients with EGID, a further peripheral blood sample was collected after an 8-week course of therapy, that is, swallowed fluticasone in 2 esophagitis cases, oral beclomethasone dipropionate in 5 colitis cases, and oral prednisone in 3 gastroenteritis cases. Splenic function was assessed by counting pitted red cells (PRC), that is, red cells with membrane abnormalities visible under phase-interference microscopy, the so-called pits. One

P.03.5 IN VIVO FORMATION OF 8-ISO-PROSTAGLANDIN F2A AND PLATELET ACTIVATION IN INFLAMMATORY BOWEL DISEASE

differentiation of human colonic fibroblasts and epithelial cells into myofibroblasts. Methods: Chronic colonic fibrosis was induced in C57BL/6 mice by successive 2.5% (w/v) DSS administration in drinking water for 6 weeks. After 6 weeks, the therapeutic effect of GED (30mg/kg/d), daily administered by oral gavage, was assessed macroscopically (weight/length of the colon, edema, ulcers, adhesions, thickness, dilatation), histologically (inflammatory infiltrate, collagen deposition) and biologically (colonic alpha-SMA, collagen I, TGFbeta1, CTGF, Smad3 and IL-13, by immunohistochemistry and immunoblotting). Differentiation of fibroblasts and intestinal epithelial cells (IECs) into myofibroblasts was induced by 4 day of TGFbeta administration (1 ng/mL and 10 ng/mL, respectively). Expression of aplha-SMA, fibronectine and the specific IECs markers (A33 and cytokeratin) was evaluated by quantitative RT-PCR. In vitro collagen deposition was observed by Picrosirius red staining. Results: In DSS-treated mice GED induced a significant 26% decrease of the colon weight/length ratio (p < 0.05) and ameliorated macroscopic and microscopic lesions determining 35% reduction of total macroscopic score (p < 0.05) and 91% (p < 0.01) of histological score. GED regulated the increased DSS-induced tissue expression of specific fibrotic markers, mainly Collagen I-III (48% p < 0.05), CTGF (35%, p < 0.05) and IL-13 (50%, p < 0.01). GED reduced mRNA expression of differentiation markers of fibroblasts and IECs, alpha-SMA (18%, p < 0.05 and 16%, p < 0.05, respectively) and fibronectine (45%, p < 0.005 and 67%, p < 0.005, respectively) and restored specific IECs markers. GED determined a 65% reduction of TGFbetainduced collagen deposition (p < 0.05), both in fibroblast and IECs. Conclusions: GED ameliorates the intestinal fibrosis in a DSS-induced chronic colitis in mice by controlling the main molecular and cellular mechanisms involved.

P.03.9 FUNCTIONAL POLYMORPHISMS OF THE RECEPTOR FOR THE ADVANCED GLYCATION END PRODUCT (RAGE) PROMOTER GENE INFLUENCE SUSCEPTIBILITY TO INFLAMMATORY BOWEL DISEASE (IBD)

FUNCTIONAL POLYMORPHISMSOF THE RECEPTOR FOR THE ADVANCED GLYCATION END PRODUCT (RAGE) PROMOTER GENE INFLUENCE SUSCEPTIBILITY TO INFLAMMATORY BOWEL DISEASE (IBD) R. Ciccocioppo∗ ,1, V. Imbesi 1, E. Betti 1 , A. Gallia 1 , V. Boccaccio1 , S. Bozzini 2 , C. Falcone2 , M. Guerci 1, A. Di Sabatino1 , G.R. Corazza1 11Clinica Medica I, Fondazione IRCCS Policlinico San Matteo, Universita degli Studi di Pavia, Pavia, Italy; 22Centro Interdipartimentale per la Ricerca in Medicina Molecolare e Sezione di Cardiologia, Istituto di Cura Citta di Pavia; Universita degli Studi di Pavia, Pavia, Italy

P.03.4 SERUM HEPCIDIN IN INFLAMMATORY BOWEL DISEASES

20)-untreated) rats. 2) TNBS up-regulated mRNA expression of COX-2, TGF-β, TNF-α, HB-EGF and t-TG in colonic tissue. 3) both concentrations of Hp 2-20 significantly (p<0.05) decreased TNBS-induced mRNA overexpression of COX-2, TNF-α, t-TG and TGF-β, whereas HB-EGF mRNA tissue levels remained unchanged; 4) TNBS up-regulated FPR and FPRL-1 mRNA expression and this was counteracted by Hp(2-20). Conclusions: 1) Peptide Hp(2-20) significantly accelerates mucosal healing in a rat model of TNBS-induced colitis, both at the macroscopic and histological level; 2) This effect is associated with a significant reduction in mRNA colonic tissue levels of COX-2, TGF-β, TNF-α, and tissue transglutaminase; 3) Hp(2-20) decreased FPR and FPRL-1 gene expression induced by TNBS; 4) We postulate that Hp(2-20) down-regulation of pro-inflammatory responses in TNBS-colitis may partly explain the inverse association between H. pylori infection and IBD.

Dosaggio dell’epcidina nel siero di pazienti con malattia infiammatoria intestinale

L’anemia e tra le piu frequenti manifestazioni extraintestinali nei pazienti affetti da malattia infiammatoria intestinale. La diagnosi differenziale tra anemia da carenza di ferro e da infiammazione cronica in questi pazienti assume un’importanza fondamentale nella scelta terapeutica; puo risultare altresi particolarmente difficoltosa, poiche spesso entrambi i meccanismi patogenetici contribuiscono all’anemizzazione nel medesimo paziente. In questo lavoro abbiamo dosato l’epcidina nel siero di pazienti affetti da malattie infiammatorie intestinali ed in un gruppo di soggetti di controllo, sani o con anemie non infiammatorie ne secondarie ad insufficienza renale. Abbiamo quindi analizzato i fattori che influenzano i livelli sierici di epcidina nelle malattie infiammatorie intestinali e la possibilita di utilizzare l’epcidina per discriminare i casi di anemia da infiammazione cronica dai casi di anemia da carenza di ferro associata ad anemia da infiammazione cronica. Tramite la concentrazione dell’epcidina e stato possibile discriminare la maggior parte dei nostri pazienti con anemia da carenza di ferro dai pazienti con anemia da infiammazione cronica, per cui possiamo assumere che il dosaggio dell’epcidina puo essere utile per una corretta diagnosi differenziale della patogenesi dell’anemia nelle malattie infiammatorie intestinali e, potenzialmente, in altre malattie infiammatorie croniche.

P037 Is ulcerative colitis an atypical Th2-mediated disease characterised by excess production of interleukin-13?

Reference(s) [1] Atreya, R.; Neurath, M. F. (2005), Involvement of IL-6 in the pathogenesis of inflammatory bowel disease and colon cancer, Clin Rev Allerg Immu [2] Galvez-Llompart, M.; Recio, M. C.; Garcia-Domenech, R. (2011), Topological virtual screening: a way to find new compounds active in ulcerative colitis by inhibiting NF-kappaB., Mol. Divers [3] Recio, M. C.; Galvez-Llompart, M.; Zanni, R; GarciaDomenech, R. (2011), New Inhibitors of Il-6 Production In Caco-2 Cells Through Molecular Topology Methodology, XXXIII Congreso de la Sociedad Espanola de Farmacologia, Malaga (Spain).