Konstantinos Arvanitidis

Low expression of interferon regulatory factor-1 and identification of novel exons skipping in patients with chronic myeloid leukaemia

Summary. Chronic myeloid leukaemia (CML) is a malignant clonal disorder of the haematopoietic stem cell. Treatment of CML patients with interferon alpha (IFN‐α) has induced haematological and cytogenetic remission. Interferons transcriptionally activate target genes through the JAK–STAT and interferon regulated factors (IRFs) family pathways. Interferon regulated factor‐1 (IRF‐1) is a transcriptional activator of genes critical for cell growth, differentiation and apoptosis. The skipping of exons 2 or 2 and 3 of IRF‐1 in patients with myelodysplastic syndromes and acute myelogenous leukaemia suggests that this factor may have a critical role in leukaemogenesis. The role of IRF‐1 in CML is currently unknown. Therefore, mutational analysis of IRF‐1 was performed and its expression pattern was also studied in CML patients. We studied IRF‐1 in peripheral blood mononuclear cells of 21 patients in chronic phase CML. No point mutations were identified at the cDNA level. Surprisingly, fourfold reduction of full‐length IRF‐1 mRNA expression was established in 17/21 patients compared with normal individuals. Low expression of full‐length IRF‐1 was observed in conjunction with high levels of aberrantly spliced mRNAs, reported for the first time. In three patients who were also analysed during blastic transformation, further reduction of full‐length IRF‐1 mRNA was observed. These findings demonstrate that, in CML patients, IRF‐1 can produce high levels of aberrant spliced mRNAs with subsequent reduction in the levels of full‐length IRF‐1 mRNA. This observation is consistent with the notion that exon skipping may constitute another mechanism of tumour suppressor gene inactivation in this disease.

Intraperitoneal application of phospholipids for the prevention of postoperative adhesions: a possible role of myofibroblasts

BACKGROUND Peritoneal adhesions, organized as fibrous bands after abdominal surgery, are related with considerable morbidity and repeated hospitalization. Phospholipids, natural constituents of the peritoneal fluid, seem to display excellent antiadhesive properties. The aim of this study was to investigate whether intraperitoneal application of phospholipids is capable of reducing postoperative adhesions and the possible underlying mechanisms. MATERIALS AND METHODS Twenty male Wistar rats were subjected to a midline laparotomy and a standard peritoneal and cecum abrasion trauma. Before laparotomy closure, a bolus of 3 mL of phospholipids (12 mg/mL) or NaCl (placebo) was given intraperitoneally. Seven days later, the quality and the quantity of adhesions, as well as serum proinflammatory and/or profibrotic mediators, were blindly assessed. Human colonic subepithelial myofibroblasts were isolated from normal controls and cultured with transforming growth factor-β1 (TGFβ1, 5 ng/mL) in the presence of phospholipids (30-300 μg/mL). Collagen production in culture supernatants and migratory activity of myofibroblasts were also assessed. RESULTS Phospholipids reduced intra-abdominal adhesions (P < 0.001), with respect to their intensity and area, and serum levels of cytokines (interleukin 1β, interleukin 6, platelet-derived growth factor-1, and TGFβ1) compared with placebo-treated rats. Stimulation of myofibroblasts with TGFβ1 significantly increased (P < 0.001) the basic collagen production. The presence of phospholipids significantly reduced (P < 0.001) both the TGFβ1 induced and the basic collagen production. Using a wound healing assay, phospholipids were found to reduce the basic and the TGFβ1-induced migration of myofibroblasts in a concentration-dependent manner. CONCLUSIONS Intraperitoneal phospholipids might be involved in the prevention of postoperative adhesions formation via the reduction of proinflammatory and/or profibrotic mediators and by inhibiting fibrogenic properties of mesenchymal cells.

Crohn's disease-associated mucosal factors regulate the expression of TNF-like cytokine 1A and its receptors in primary subepithelial intestinal myofibroblasts and intestinal epithelial cells.

&NA; Intestinal subepithelial myofibroblasts (SEMFs) exert a profibrotic role in Crohns disease (CD). Tumor necrosis factor‐like cytokine 1A (TL1A) and its receptors, death‐domain receptor 3 (DR3) and decoy receptor 3 (DcR3), are mucosal factors with significant involvement in experimental inflammation and CD. We aimed to determine the regulation of expression of this system of proteins in SEMFs and intestinal epithelial cells. The relative amount of mRNA transcripts for TL1A, DR3, and DcR3 was measured by real‐time reverse transcription polymerase chain reaction in cultured primary SEMFs, colonic myofibroblast cell line 18CO, and epithelial cell line HT29. Protein expression was determined by immunofluorescence. The effect of various proinflammatory stimuli in mRNA and protein expression was studied. TL1A mRNA and protein expression in primary SEMFs (and 18CO cells) was significantly upregulated after stimulation with interleukin 1‐alpha and/or tumor necrosis factor alpha (TNF‐&agr;) (32‐ to 44‐fold increase, P < 0.05 vs unstimulated). Following stimulation with interleukin 1‐alpha + TNF‐&agr; + IFN‐&ggr;, HT‐29 cells highly expressed DR3 (4.1‐fold over unstimulated, P = 0.008) and DcR3 (56‐fold, P = 0.009) and secreted soluble factors that led to induction of TL1A mRNA in primary SEMFs (28‐fold, P = 0.008). Activated epithelial cells significantly upregulated IL‐8 expression in response to stimulation with recombinant TL1A. Supernatants from mucosal cultures of patients with CD were able to stimulate the expression of TL1A in cultured primary SEMFs, in comparison to supernatants from healthy controls (3.8‐fold increase, P < 0.05) or culture media alone (P < 0.05). In conclusion, we found that proinflammatory cytokines are important regulators of the expression of TL1A in SEMFs and of its receptors in intestinal epithelial cells. Our results raise the possibility for involvement of TL1A/DR3/DR3‐mediated mechanisms in epithelial‐mesenchymal interactions and the development of inflammation‐induced intestinal fibrosis in CD.

Combined Enteral and Parenteral Glutamine Supplementation in Endotoxaemic Swine: Effects on Portal and Systemic Circulation Levels

Objective: To measure plasma glutamine (GLN) levels in systemic and portal circulation after combined enteral and parenteral administration in early endotoxemic swine. We hypothesized that this combination will be more efficient than intravenous administration alone in restoring plasma levels during the course of endotoxemia. Materials and Methods: Endotoxemia was induced with Escherichia coli O111:B4 lipopolysaccharide (LPS) (250 μg/kg body weight) in 16 anesthetized, fasted swine and maintained by constant infusion (2 μg/kg/h) over 180 min. Another 16 swine served as controls. After infusion with LPS or placebo, GLN was administered intravenously, enterally or in combination (0.5 g/kg i.v. plus 0.5 g/kg enterally) over 30 min. At 0, 15, 30, 45, 60, 120 and 180 min, blood was drawn from the systemic and portal circulation for colorimetric assessment of GLN. Results: In healthy, placebo-alone swine, GLN levels remained stable throughout the study. Intravenous and combined infusion increased systemic levels (p = 0.001), but after enteral administration alone, a smaller effect was observed (p = 0.026). Portal levels were increased after combined, enteral and intravenous administration (p = 0.001). In endotoxemia, systemic and portal levels decreased significantly. Intravenous and, to a greater extent, combined administration increased systemic levels (p = 0.001), while enteral administration only had a small effect (p = 0.001). In the portal vein, intravenous and combined treatment increased plasma levels (p = 0.001), whereas enteral supplementation alone had again a small, yet significant effect (p = 0.001). Conclusions: The findings indicate that combined GLN supplementation is superior to intravenous treatment alone, in terms of enhanced availability in systemic and portal circulations. Thus, combined treatment at the onset of endotoxemia is a beneficial practice, ensuring adequate GLN to compensate for the resulting intracellular shortage.

P032 Pro-inflammatory cytokines induce the expression of TL1A/TNFSF15 in primary intestinal subepithelial myofibroblasts

P032 Pro-inflammatory cytokines induce the expression of TL1A/TNFSF15 in primary intestinal subepithelial myofibroblasts G. Bamias1 *, E. Filidou2, D. Goukos3, V. Valatas4, K. Arvanitidis2, M. Panagopoulou2, G. Kouklakis5, G. Daikos3, S. Ladas1, G. Kolios2. 1Laikon Hospital, Ethnikon & Kapodistriakon University of Athens, Academic Dpt. of Gastroenterology, Athens, Greece, 2Democritus University of Thrace, Laboratory of Pharmacology, Faculty of Medicine, Alexandroupolis, Greece, 3Laikon Hospital, Ethnikon & Kapodistriakon University of Athens, 1st. Dpt of Propaedeutic and Internal Medicine, Laboratory of Infectious Diseases, Athens, Greece, 4University of Crete, Laboratory of Gastroenterology, Faculty of Medicine, Heraklion, Greece, 5Democritus University of Thrace, Endoscopy Unit, Faculty of Medicine, University Hospital of Alexandroupolis, Alexandroupolis, Greece