Pinelopi Manousou

Prognostic models in cirrhotics admitted to intensive care units better predict outcome when assessed at 48 h after admission

Background and Aim:  The accuracy of prognostic models in critically ill cirrhotics at admission to intensive care units (ICU) may be unreliable. Predictive accuracy could be improved by evaluating changes over time, but this has not been published. The aim of the present study was to assess the performance of prognostic models in cirrhotics at admission (baseline) and at 48 h to predict mortality in the ICU or within 6 weeks after discharge from the ICU.

Secretion of inflammatory mediators by isolated rat Kupffer cells: the effect of octreotide

AIMS We studied the production of inflammatory mediators by rat KC and the possible in vitro effect of the somatostatin analogue octreotide. METHODS Primary KC cultures were incubated with LPS added alone or with different concentrations of octreotide. The production of TNFalpha, IL-6, IL-10, IL-12 and IL-13 was assessed in culture supernatants by ELISA and that of nitric oxide (NO) by a modification of the Griess reaction. RESULTS Isolated KC produced a basal amount of TNFalpha, IL-6, IL-12, IL-13, and NO but not IL-10. LPS-stimulated KC secreted significantly increased amounts of TNFalpha (P < 0.001), IL-6 (P < 0.01), IL-10 (P < 0.001), IL-12 (P < 0.01), and NO (P < 0.001) whereas IL-13 production remained constant. Octreotide reduced IL-12 (P < 0.05) and increased IL-13 (P < 0.05) production by unstimulated KC. Furthermore, octreotide suppressed TNFalpha production (P < 0.05), without modifying TNFalpha mRNA expression and decreased iNOS expression and NO (P approximately 0.05) production by LPS-activated KC. These effects were reversed with Wortmannin pre-treatment suggesting that octreotide may act via interference with phosphatidylinositol 3-kinase pathways. CONCLUSIONS These data demonstrate that KC is a source of multiple inflammatory mediators, indicating a critical role in liver inflammatory disorders. Octreotide modulates inflammatory mediator production by isolated KC, suggesting that it might have immunoregulatory and anti-inflammatory effects in liver diseases.

Octreotide regulates CC but not CXC LPS-induced chemokine secretion in rat Kupffer cells

Kupffer cells (KC) and lipopolysaccharide (LPS) interaction is the initial event leading to hepatic inflammation and fibrosis in many types of liver injury. We studied chemokine secretion by KC activated with LPS and the possible effect of the somatostatin analogue octreotide, in the regulation of this process. KC isolated from Sprague–Dawley rats were cultured in the presence of LPS added alone or with different concentrations of octreotide for 24 and 48 h, and chemokine production was assessed in culture supernatants by ELISA. CC chemokine mRNA expression was assessed by semiquantitative RT–PCR. Vehicle‐stimulated KC produced a basal amount of CC and CXC chemokines. LPS‐stimulated KC secreted significantly increased amounts of IL‐8 (GRO/CINC‐1) (P<0.001), MIP‐2 (P<0.001), MCP‐1 (P<0.001), and RANTES (P<0.01). Octreotide inhibited LPS‐induced secretion of the CC chemokines MCP‐1 (P<0.05) and RANTES (P<0.05), but not the CXC chemokines IL‐8 (GRO/CINC‐1) and MIP‐2, in a concentration‐dependent manner. Downregulation of basal and LPS‐induced mRNA expression of the CC chemokines was also observed in the presence of octreotide. Pretreatment with phosphatidylinositol 3 (PI3)‐kinase inhibitors reduced chemokine production by LPS‐treated KC in both the mRNA and protein level. Furthermore, it prevented the octreotide inhibitory effect on LPS‐induced chemokine secretion, indicating a possible involvement of the PI3‐kinase pathway. In conclusion, these data demonstrate that chemokine secretion by KC can be differentially regulated by octreotide, and suggest that this somatostatin analogue may have immunoregulatory effects on resident liver macrophages.

Proinflammatory cytokines induce crosstalk between colonic epithelial cells and subepithelial myofibroblasts: Implication in intestinal fibrosis

BACKGROUND AND AIMS Colonic epithelial cells and adjacent subepithelial myofibroblasts are important counterparts in the pathogenesis of intestinal inflammation and fibrosis. We investigated the possible crosstalk between them, whilst focusing on the mucosal inflammation pathways that potentially trigger intestinal fibrosis. METHODS We studied the effects of proinflammatory cytokines (IL-1α, TNF-α, IFN-γ) on human colonic epithelial cell lines and the effects of epithelial cell-conditioned media on primary human colonic subepithelial myofibroblasts isolated from normal controls or patients with inflammatory Crohns disease along with the corresponding 18CO cell line. Readouts included production of TGF-β and TIMP-1, total collagen synthesis, matrix metalloproteinases MMP-2 and MMP-9 and myofibroblast migration/mobility. RESULTS Proinflammatory cytokines upregulated TGF-β and TIMP-1 in colonic epithelial cells. Conditioned medium from these epithelial cell cultures induced production of MMP-9 and collagen and inhibited the migration/mobility of subepithelial myofibroblasts. MMP-9 production depended on endothelin receptor A signalling on responding myofibroblasts. Collagen up-regulation was independent of TGF-β, CTGF, TF and endothelin. Subepithelial myofibroblasts isolated from Crohns disease patients had similar responses to those isolated from normal controls, with the exception of higher basal collagen production. CONCLUSIONS Our study indicates that colonic epithelial cells may respond to an inflammatory milieu by inducing myofibroblast functions similar to those observed during intestinal fibrosis.

Production of pro- and anti-fibrotic agents by rat Kupffer cells; the effect of octreotide.

Kupffer cells may be involved in liver fibrogenesis through production of TGF-β1. Their role in fibrinolysis is less clear. Octreotide, a synthetic analogue of somatostatin, is often used in cirrhotic patients. Its effect on Kupffer cells was studied. Isolated rat Kupffer cells were cultured in the presence of lipopolysaccharide and/or octreotide. TGF-β1, leptin, collagenase (MMP-1), and urokinase-type plasminogen activator (uPA) were assessed in supernatants by ELISA, and MMP-2 and MMP-9 by zymography. Kupffer cells produced large amounts of MMP-1 and lipopolysaccharide induced a significant (P < 0.02) early increase. Octreotide and lipopolysaccharide caused a synergistic effect on MMP-1 secretion. By contrast, MMP-9 production stimulated by lipopolysaccharide was suppressed by octreotide. Kupffer cells produced a basal amount of uPA, significantly increased after lipopolysaccharide or octreotide incubation (P < 0.001). Large amounts of TGF-β1 were produced in a time-dependent manner by unstimulated Kupffer cells. Lipopolysaccharide and octreotide, alone or in combination, induced a significant inhibition of this production (P < 0.01). Kupffer cells did not produce leptin, a recently identified mediator of liver fibrosis, or MMP-2. Kupffer cells may play a significant role in liver fibrinolysis. Octreotide, acting on TGF-β1, uPA, and MMP-1 production, may be a useful agent for fibrosis resolution.

Increased expression of chemokine receptor CCR3 and its ligands in ulcerative colitis: the role of colonic epithelial cells in in vitro studies

Human colonic epithelial cells express T helper type 1 (Th1)‐associated chemoattractants, yet little is known about the production of Th2‐associated chemoattractants. CCL11/eotaxin‐1, CCL24/eotaxin‐2 and CCL26/eotaxin‐3 are known to attract CCR3‐expressing, Th2‐polarized lymphocytes. We studied constitutive and inflammation‐induced expression and production of CCR3 together with its ligands in the colon and peripheral blood of patients with inflammatory bowel disease (IBD) by flow cytometry, reverse transcription–polymerase chain reaction (RT–PCR) and enzyme‐linked immunosorbent assay (ELISA). We further defined the regulated expression of these chemokines by RT–PCR and ELISA using cultured human epithelial cell lines. A higher fraction of peripheral T lymphocytes were found to be positive for CCR3 in patients with ulcerative colitis (UC) compared to Crohns disease (CD), while almost no CCR3+ T cells were found in normal controls (NC). Similarly, higher and more frequent expression of CCR3 was observed in colonic biopsies from patients with UC, regardless of the disease activity, when compared to CD or NCs. Serum CCL11/eotaxin‐1 was increased significantly in UC (306 ± 87 pg/ml) and less so in CD (257 ± 43 pg/ml), whereas CCL24/eotaxin‐2, and CCL26/eotaxin‐3 were increased only in UC. Colonic expression of the three chemokines was minimal in NCs but high in inflammatory bowel diseases (especially UC) and was independent of disease activity. Th2, and to a lesser extent Th1, cytokines were able to induce expression and production of all three eotaxins from colonic epithelial cells in culture. CCR3 and ligands over‐expression would appear to be a characteristic of UC. The production of CCR3 ligands by human colonic epithelial cells suggests further that epithelium can play a role in modulating pathological T cell‐mediated mucosal inflammation.

Oxidative stress due to anesthesia and surgical trauma: Importance of early enteral nutrition

Anesthesia and surgical trauma are considered major oxidative and nitrosative stress effectors resulting in the development of SIRS. In this study we evaluated the usefulness of early enteral nutrition after surgical trauma. Sixty male Wistar rats were subjected to midline laparotomy and feeding-gastrostomy. Twenty of these rats served as controls after recovering from the operation stress. The remaining rats received, through gastrostomy, enteral nutrition or placebo-feeding for 24 h. Oxidative stress markers and CC chemokine production were evaluated in rat serum and liver tissue. The operation itself was found to increase nitric oxide (NO) and malondialdehyde (MDA) and to decrease superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), as well as liver tissue energy charge (EC) in relation to controls. The rats receiving enteral feeding exhibited statistically significantly lower levels of NO and MDA, and higher levels of SOD, GSH-Px, and liver EC, in relation to placebo feeding rats. The operation significantly increased the chemokines monocyte chemoattractant protein (MCP)-1 and regulated upon activation, normal T-cell expressed, and secreted (RANTES) in rat serum, while enteral nutrition caused a further significant increase in chemokine levels in serum. mRNA chemokine expression in liver was increased in a similar pattern. These findings indicate that early enteral feeding might play an important role after surgery ameliorating oxidative stress, affecting positively the hepatic EC and regulating, via chemokine production, cell trafficking, and healing process.

Ciprofloxacin inhibits cytokine‐induced nitric oxide production in human colonic epithelium

Background  The fluoroquinolone ciprofloxacin is a broad‐spectrum antibiotic that has been used in the treatment of inflammatory bowel diseases. There is evidence that quinolones have immunomodulating activities via the regulation of cytokine production.

Ciprofloxacin decreases survival in HT-29 cells via the induction of TGF-β1 secretion and enhances the anti-proliferative effect of 5-fluorouracil

Background and purpose:  Fluoroquinolones are potent anti‐microbial agents with multiple effects on host cells and tissues. Previous studies have highlighted their pro‐apoptotic effect on human cancer cells and an immunoregulatory role in animal models of inflammatory bowel disease. We examined the effect of ciprofloxacin on proliferation, cell cycle and apoptosis of HT‐29 cells, a human colonic epithelial cell line sensitive to transforming growth factor (TGF)‐β1‐mediated growth inhibition and its role in TGF‐β1 production. We also examined the effect of ciprofloxacin on proliferation of HT‐29 cells in combination with 5‐fluorouracil (5‐FU), a well‐established pro‐apoptotic agent.

Expression of a splice variant of CXCR3 in Crohn's disease patients; indication for a lymphocyte—epithelial cell interaction

Background and Aim:  T‐lymphocyte migration is implicated in the pathogenesis of Crohns disease (CD) and ulcerative colitis (UC). CXC chemokines MIG, IP‐10, and I‐TAC act by binding to CXCR3 receptor on T‐lymphocytes. We investigated the role of these chemokines and their receptor in patients with UC, CD, and normal controls (NC).