Costas Xidakis

Circulating levels of leptin, adiponectin, resistin, and ghrelin in inflammatory bowel disease

Background: There is evidence that adipocytokines play an important role in metabolism and in inflammation. Because human metabolism dramatically changes in inflammatory bowel disease (IBD) and chronic inflammation is the hallmark of the disease, we studied serum levels of leptin, adiponectin, resistin, and ghrelin in patients with ulcerative colitis (UC) and Crohns disease (CD) in comparison with healthy controls (HC). Methods: Leptin, adiponectin, resistin, and active ghrelin serum levels were measured in 100 IBD patients (46 UC and 54 CD) and in 60 matched HC using commercially available enzyme‐linked immunosorbent assays. Leptin, adiponectin, resistin, and ghrelin levels were correlated with disease activity, type, localization, and treatment. Results: Mean serum leptin levels were 10.6 ± 2.0 ng/mL in UC patients, 12.5 ± 2.6 ng/mL in CD patients, and 15.0 ± 1.8 ng/mL in HC (P = .01). Mean serum adiponectin levels were 9514.8 ± 787.8 ng/mL in UC patients, 7651.1 ± 613 ng/mL in CD patients, and 7270.6 ± 559.4 ng/mL in HC (P = .05). Mean serum resistin levels were 21.2 ± 2.2 ng/mL in UC patients, 18.7 ± 1.6 ng/mL in CD patients and 11.8 ± 0.6 ng/mL in HC (P = .0002). Mean serum ghrelin levels were 48.2 ± 4.2 pg/mL in UC patients, 49.4 ± 4.6 pg/mL in CD patients and 14.8 ± 3.0 pg/mL in HC (P < .0001). Serum levels of these adipocytokines were not correlated with either C‐reactive protein levels or the clinical indices of activity. No association between serum adipocytokines levels and disease localization in both UC and CD patients was found. Only serum ghrelin was significantly higher in ileal compared with colonic CD (P = .04). Conclusions: Serum levels of adiponectin, resistin, and active ghrelin are increased whereas serum levels of leptin are decreased in patients with IBD. Further studies are needed to elucidate the role of adipocytokines in IBD.

Isolation of rat Kupffer cells: a combined methodology for highly purified primary cultures.

We report a four‐step procedure that optimizes the methodology for isolation of highly purified rat Kupffer cells (KC). We combined the previously reported techniques of enzymatic tissue treatment, density gradient centrifugation, centrifugal elutriation and selective adherence. ED‐2 immunophenotyping and non‐specific esterase histochemistry were used for cell identification. This combination resulted in a satisfactorily high yield of 80–100×106KCs per liver, over 95% positive for ED‐2 and 98% viable cells. Cultures of isolated KCs were functionally intact and exhibited a concentration and time‐dependent LPS‐induced TNF‐α and nitric oxide production.

Effectiveness of darbepoetin-alfa in combination with intravenous iron sucrose in patients with inflammatory bowel disease and refractory anaemia : a pilot study

Background The combination of intravenous iron and recombinant human erythropoietin has been proved to be effective in the treatment of refractory anaemia in patients with inflammatory bowel disease (IBD). Darbepoetin-alpha (DPO) has a three-fold longer terminal half-life than erythropoietin. The purpose of this pilot study was to determine whether darbepoetin-alpha is also effective for the treatment of refractory anaemia in IBD. Methods Twenty IBD patients (nine ulcerative colitis and 11 Crohns disease) and refractory anaemia received intravenous iron sucrose (total iron dose 1.3±0.5 g, range 0.7–1.9) and darbepoetin-alfa at the single, weekly dose of 0.9 μg/kg subcutaneously for 4 weeks. Serum erythropoietin, ferritin, transferrin, soluble transferrin receptor, C-reactive protein and interleukin-6 were measured at baseline and after treatment. Results Haematopoietic response (increase of haemoglobin ≥2.0 g/dl) was observed in 15 out of the 20 patients (75%). The mean haemoglobin concentrations increased from 9.48±0.82 g/dl at baseline to 12.71±1.12 g/dl after treatment (P<0.0001). Mean corpuscular volume and serum ferritin levels were also significantly increased whereas mean C-reactive protein levels and endogenous erythropoietin levels significantly decreased after treatment. Conclusions In IBD patients with refractory anaemia the administration of darbepoetin in combination with intravenous iron sucrose can raise haemoglobin levels.

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.

Apoptosis and apoptosis related proteins in chronic viral liver disease

AbstractBackground: Apoptosis may be an important mechanism of hepatocyte death in chronic viral liver disease. Methods: We studied apoptosis in liver biopsies from 30 patients with chronic viral hepatitis and 8 patients with viral cirrhosis by the TUNEL method. 12 cases of non-alcoholic steatohepatitis and 12 cases of primary biliary cirrhosis were used as non-viral disease controls. Immunohistochemical expression of p53, p21/waf1, bcl-2 and mdm-2 proteins was also studied in the same patients. Results: A statistically significant increase of apoptotic liver cells was found in severe chronic viral hepatitis (5.3 ± 0.3%), cirrhosis (3.4 ± 0.5%) and PBC (4.4 ± 0.4%) cases compared to patients with non-alcoholic steatohepatitis (0.8 ± 0.3%). The expression of p53 protein was increased in the cases of viral cirrhosis and in chronic severe viral hepatitis whereas in the cases of chronic mild hepatitis, PBC and non-alcoholic steatohepatitis we found no expression of p53. P21/waf1 expression was increased in severe chronic hepatitis, cirrhosis and PBC cases compared to mild hepatitis and non-alcoholic steatohepatitis cases. However no induction of mdm-2 was observed in the subgroups of chronic liver disease. Bcl-2 was expressed only in epithelium of bile ducts and mononuclear cells of the portal tracts and liver lobules. A weaker Bcl-2 expression was noted in the epithelium of bile ducts of 7/12 PBC cases. Conclusion: Our results provide evidence of increased apoptosis in severe chronic viral liver disease, suggesting that apoptotic cell death might be involved in the pathogenesis of hepatocellular damage of viral hepatitis and cirrhosis. Furthermore we analysed part of the apoptotic pathways implicated in the above process and found an increased expression of p21/waf1, probably p53 mediated, without overexpression of the apoptosis inhibiting bcl-2 and mdm-2 proteins. By contrast p21/waf1 overexpression in PBC seems to be propagated by a p53 independent mechanism.

Association between enhanced soluble CD40 ligand and prothrombotic state in inflammatory bowel disease.

Background Inflammatory bowel disease is associated with an increased incidence of thromboembolic complications. The aim of this study was to investigate the role of the soluble CD40 ligand (sCD40L), which displays prothrombotic properties, in patients with ulcerative colitis (UC) and Crohns disease (CD) in comparison with inflammatory and healthy controls. Methods Plasma levels of sCD40L, prothrombin fragment 1+2 (F1+2), thrombin–antithrombin (TAT) complex and soluble P-selectin were measured in 104 inflammatory bowel disease patients (54 ulcerative colitis and 50 Crohns disease), in 18 cases with other causes of intestinal inflammation and in 80 healthy controls using commercially available enzyme-linked immunosorbent assays. Plasma levels of sCD40L were correlated with disease activity, type, localization and treatment as well as with the measured thrombophilic parameters. Results CD patients had significantly higher sCD40L levels than both groups of controls (CD vs HC P < 0.001; CD vs non-IBD P < 0.05). UC patients had higher but not significantly different sCD40L levels compared with the controls. Both UC and CD patients with active disease had significantly higher sCD40L levels in comparison with patients with inactive disease. Plasma levels of sCD40L were correlated with platelet count (r = 0.27, P = 0.001). They also showed a correlation with prothrombin F1+2 (r = 0.16, r = 0.03) and TAT (r = 0.15, r = 0.04) as well as with P-selectin (r = 0.19, P = 0.01). Conclusions The increased sCD40L plasma levels may represent, at least in some degree, a molecular link between inflammatory bowel disease and the procoagualant state.

Serum Angiogenin in Inflammatory Bowel Disease

Angiogenesis-promoting cytokines have been suggested to play an important role in inflammatory bowel disease (IBD) since they promote inflammation by increasing vascular permeability and mediate tissue repair by activating fibroblasts. The aim of the present study was to evaluate the serum levels of angiogenin, a potent angiogenic factor, in patients with ulcerative colitis (UC) and Crohn’s disease (CD). Angiogenin serum levels were measured in 154 IBD patients (78 UC and 76 CD), in 18 cases with other causes of intestinal inflammation, and in 84 matched healthy controls using a commercially available enzyme-linked immunosorbent assay. Angiogenin levels were assessed in terms of disease activity, type, localization, and treatment. Mean (±SD) serum angiogenin levels were 526.5 ± 224.1 ng/ml in UC patients, 508.8 ± 228.5 ng/ml in CD patients, 394.6 ± 137.6 ng/ml in healthy controls, and 448.1 ± 167.8 ng/ml in patients with non-IBD intestinal inflammation. A statistically significant difference among the mean levels of angiogenin in the four groups was found (P=0.0003). IBD patients with early disease had a significantly lower mean serum angiogenin compared with patients with late disease (P=0.03). No significant association between angiogenin levels and disease activity, localization, disease type, or treatment was found. Serum angiogenin is elevated in patients with IBD. The increased serum angiogenin suggests that angiogenin may mediate angiogenesis and vascular permeability in the mucosa of patients with IBD.

The effect of infliximab on circulating levels of leptin, adiponectin and resistin in patients with inflammatory bowel disease.

Background Tumour necrosis factor &agr; is a critical mediator of inflammation-related altered metabolism in inflammatory bowel disease (IBD), possibly through its interaction with adipokines, which play an important role in IBD. Infliximab is a well established antitumour necrosis factor &agr; treatment in IBD. Aim and methods We studied serum levels of leptin, adiponectin and resistin in 20 IBD patients before and after infliximab treatment using commercially available enzyme-linked immunosorbent assays. The results were correlated with alterations of disease activity, BMI and C-reactive protein. Results Infliximab induced clinical response or remission in 18 out of 20 treated IBD patients. Mean serum-leptin levels were 4.6±0.5 and 5.1±0.5 ng/ml (P=0.41), mean serum-adiponectin levels were 10513.9±1216.9 and 9653.5±1031.5 ng/ml (P=0.36) and mean serum-resistin levels were 26.3±4.1 and 13.9±1.4 ng/ml (P=0.004), before and after infliximab treatment, respectively. No significant correlation between the changes of BMI, C-reactive protein or the clinical indices of activity and alterations of the examined adipokines was found. Conclusions Serum levels of leptin and adiponectin had no significant alterations, whereas serum-resistin levels are significantly decreased after infliximab therapy in IBD patients, suggesting a possible proinflammatory status for resistin in IBD and a role as a marker of successful therapy.

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.