- Email: [email protected]
554 EFFECTS OF URSODEOXYCHOLIC ACID AND L-ORNITHINEL- ASPARTATE ON HEPATOCYTES CHANGES IN ALD PATIENTS
POSTERS multiple organ failure with high mortality. Neutrophils are abundant innate immune cells and function via controlled release of their primary, secondary and secretory granule subsets with specific targeted functions. The aim of this study was to characterise neutrophil granule phenotype and function, comparing patients with ALD/AAH against healthy controls (HC) to interrogate whether granule dysfunction contributes to the susceptibility to infection. Methods: A case–control study was undertaken comparing peripheral blood neutrophils from patients with advanced [Child Pugh B/C] ALD (n = 15) and AAH (n = 5) against HC (n = 7). Peripheral whole blood was stained with fluorochrome-labelled antibodies directed against specific neutrophil granule proteins both intracellularly and extracellularly [CD11b (secretory granules), CD63 and myeloperoxidase (primary granules), and CD66b (secondary granules)], pre- and post-stimulation with fMLP (Nformyl-methionine-leucine-phenylalanine) and opsonised E. Coli. Results were analysed utilising FACS Canto II flow cytometry and ImageStream flow cytometry which combines quantitative image analysis and flow cytometry in a single platform. ELISA and cytokine bead arrays were performed to quantify baseline plasma lactoferrin and cytokine profiles. Results: Circulating neutrophils from ALD patients had increased expression of the surface endothelial adhesion marker CD11b at baseline (p = 0.04) with augmented intracellular and extracellular responses to stimulation with fMLP. Primary granules were also upregulated at baseline (p = 0.04) and demonstrated exaggerated responses to stimulation (p = 0.03). ImageStream flow cytometry following incubation with FITC-labelled E. Coli confirmed robust granule co-localisation of CD11b, CD16, CD63, myeloperoxidase and CD66b with phagocytosed E. Coli similar to that of HC. Plasma lactoferrin (released from secondary granules) and interleukin-8 (potent neutrophil-attracting chemokine) were elevated across the spectrum of ALD, most marked in AAH (p = 0.04 and p = 0.001, respectively). Conclusions: Circulating neutrophils in patients with ALD/AAH are pre-primed, with increased endothelial adhesion markers and augmented intracellular granule mobilisation and extracellular release. Impaired granule mobilisation does not appear to contribute to the functional immunoparesis observed in patients with ALD/AAH but may contribute to bystander damage (myeloperoxidase) inducing systemic inflammation and organ dysfunction. 553 LEPTIN ADMINISTRATION REGULATES HEPATIC CHOLESTEROL SYNTHESIS IN A MOUSE MODEL OF ALCOHOLIC FATTY LIVER DISEASE B. Vairappan1 , M. Gopal2 , N. Namasivayam1 . 1 Department of Biochemistry and Biotechnology, Annamalai University, Chidambaram, 2 Department of Biotechnology, National Center for Cell Science, Pune, India E-mail: [email protected] Alcohol induced fatty liver disease is the most common and earliest response to the progression of fibrosis and/or cirrhosis. The mechanism by which ethanol causes fatty liver disease is complex and not fully understood, however, enhanced hepatic lipogenesis has been proposed as an important biochemical mechanism. The purpose of this study was to evaluate the effect of exogenous leptin administration on ethanol induced hepatic cholesterol synthesis in mice. Methods: CD-1 mice (n = 10/group) were studied for 45 days. Four groups were studied. 1. control, 2. leptin+control (230 mg/kg intraperitoneal every alternate day from day 15), 3. alcohol (6.32 g/kg daily by gastric lavage, for 45 days) and S226
4. alcohol plus leptin (as prior dosing). Results: Compared to control, ethanol supplementation significantly (p < 0.05) increased levels of plasma total and ester cholesterol and the activities of the enzymes HMG CoA reductase and cholesterol ester synthase (CES) which were normalized by addition of leptin (p < 0.05). Increased SREBP2 protein expression found in ethanol fed mice was also normalised by leptin treatment. The activities of hepatic lipoprotein lipase (LPL), plasma lecithin cholesterol acyl transferase (LCAT) and tissue cholesterol ester hydrolase (CEH) were significantly (p < 0.05) lowered following ethanol supplementation compared to control mice. These features were significantly increased by addition of leptin. Furthermore, significantly increased excretion of total bile acids and neutral sterols were observed on leptin administration to ethanol fed mice. Liver histology showed that mice given ethanol had macro and micro vesicular steatosis. However, ethanol + leptin treated liver showed sinusoidal dilatation and no fatty change. Leptin injection in control mice showed mild sinusoidal dilatation and normal hepatocytes. Conclusion: Thus, administration of exogenous leptin to ethanolsupplemented mice markedly decreased the synthesis and increased the catabolism of cholesterol in the liver. 554 EFFECTS OF URSODEOXYCHOLIC ACID AND L-ORNITHINEL-ASPARTATE ON HEPATOCYTES CHANGES IN ALD PATIENTS N. Virstyuk, O. Deltsova, S. Geraschenko, L. Kovalchuk. Central Hospital, Ivano-Frankivsk National Medical University, Ivano-Frankivsk, Ukraine E-mail: [email protected] Background and Aims: The aim of this study was to investigate the effects of ursodeoxycholic acid (UDCA) and L-ornithine-L-aspartate on electronomicroscopic, morphometrical and cytogenetic changes of hepatocytes in alcoholic liver disease (ALD) patients. Methods: In 48 ALD patients [age, 44.2±6.8 yr] with alcoholic hepatitis and 12 healthy volunteers [age, 44.3±5.5 yr] circulating type IV collagen and basic fibroblast growth factor (bFGF) levels, electronomicroscopic, morphometrical, cytogenetic parameters of the hepatocytes and hepatocytes nuclears were determined. 26 ALD patients treated with 10 mg/kg/day of UDCA (I group), 22 ALD patients treated with 10 mg/kg/day of UDCA+L-ornithine-Laspartate 9 g/day for six months (II group). Results: After therapy the condensed chromatin content was decreased in hepatocytes nuclear in II group (p < 0.05). The quantity of the pathologically changed nuclears was decreased more than 2.5 times and 1.8 times (p < 0.05) in II and I groups vs. patients before treatment. In II group the area of the hepatocytes profile (AHP) was increased up to (165.15±11.08) mm2 vs. (119.80±10.53) mm2 (p < 0.05); in I group – (139.65±11.53) mm2 vs. (120.07±10.26) mm2 (p < 0.05) before treatment and decreased vs. (182.17±2.54) mm2 (p < 0.05) in control. After therapy 5 (22.73%) patients in II group and 13 (46.4%) in I group have AHP from 50.0 to 150.0 mm2 vs. 18 (81.8%) and 21 (80.76%) patients before treatment; in healthy volunteers AHP was from 100.0 to 250.0 mm2 (85.42%). The area of the nuclear profile of patients in II group was increased – up to (25.28±1.84) mm2 vs. (16.96±1.02 mm2 ) (p < 0.05), in I group – (21.70±1.25) mm2 vs. (17.35±1.19 mm2 ) (p < 0.05) before treatment and decreased vs. (35.32±0.60) mm2 in control. The circulating type IV collagen levels decreased more than 1.8 times and 1.4 times (p < 0.05), bFGF levels decreased more than 2.2 times and 1.6 times (p < 0.05) in II and I groups vs. patients before treatment (P < 0.02). Conclusions: The UDCA+L-ornithine-L-aspartate combination in long-term use has antifibrotic effects with beneficial cytoprotective properties and improves electronomicroscopic, morphometrical and cytogenetic markers of the hepatocytes and hepatocytes nuclears in ALD patients.
Journal of Hepatology 2013 vol. 58 | S63–S227
4. alcohol plus leptin (as prior dosing). Results: Compared to control, ethanol supplementation significantly (p < 0.05) increased levels of plasma total and ester cholesterol and the activities of the enzymes HMG CoA reductase and cholesterol ester synthase (CES) which were normalized by addition of leptin (p < 0.05). Increased SREBP2 protein expression found in ethanol fed mice was also normalised by leptin treatment. The activities of hepatic lipoprotein lipase (LPL), plasma lecithin cholesterol acyl transferase (LCAT) and tissue cholesterol ester hydrolase (CEH) were significantly (p < 0.05) lowered following ethanol supplementation compared to control mice. These features were significantly increased by addition of leptin. Furthermore, significantly increased excretion of total bile acids and neutral sterols were observed on leptin administration to ethanol fed mice. Liver histology showed that mice given ethanol had macro and micro vesicular steatosis. However, ethanol + leptin treated liver showed sinusoidal dilatation and no fatty change. Leptin injection in control mice showed mild sinusoidal dilatation and normal hepatocytes. Conclusion: Thus, administration of exogenous leptin to ethanolsupplemented mice markedly decreased the synthesis and increased the catabolism of cholesterol in the liver. 554 EFFECTS OF URSODEOXYCHOLIC ACID AND L-ORNITHINEL-ASPARTATE ON HEPATOCYTES CHANGES IN ALD PATIENTS N. Virstyuk, O. Deltsova, S. Geraschenko, L. Kovalchuk. Central Hospital, Ivano-Frankivsk National Medical University, Ivano-Frankivsk, Ukraine E-mail: [email protected] Background and Aims: The aim of this study was to investigate the effects of ursodeoxycholic acid (UDCA) and L-ornithine-L-aspartate on electronomicroscopic, morphometrical and cytogenetic changes of hepatocytes in alcoholic liver disease (ALD) patients. Methods: In 48 ALD patients [age, 44.2±6.8 yr] with alcoholic hepatitis and 12 healthy volunteers [age, 44.3±5.5 yr] circulating type IV collagen and basic fibroblast growth factor (bFGF) levels, electronomicroscopic, morphometrical, cytogenetic parameters of the hepatocytes and hepatocytes nuclears were determined. 26 ALD patients treated with 10 mg/kg/day of UDCA (I group), 22 ALD patients treated with 10 mg/kg/day of UDCA+L-ornithine-Laspartate 9 g/day for six months (II group). Results: After therapy the condensed chromatin content was decreased in hepatocytes nuclear in II group (p < 0.05). The quantity of the pathologically changed nuclears was decreased more than 2.5 times and 1.8 times (p < 0.05) in II and I groups vs. patients before treatment. In II group the area of the hepatocytes profile (AHP) was increased up to (165.15±11.08) mm2 vs. (119.80±10.53) mm2 (p < 0.05); in I group – (139.65±11.53) mm2 vs. (120.07±10.26) mm2 (p < 0.05) before treatment and decreased vs. (182.17±2.54) mm2 (p < 0.05) in control. After therapy 5 (22.73%) patients in II group and 13 (46.4%) in I group have AHP from 50.0 to 150.0 mm2 vs. 18 (81.8%) and 21 (80.76%) patients before treatment; in healthy volunteers AHP was from 100.0 to 250.0 mm2 (85.42%). The area of the nuclear profile of patients in II group was increased – up to (25.28±1.84) mm2 vs. (16.96±1.02 mm2 ) (p < 0.05), in I group – (21.70±1.25) mm2 vs. (17.35±1.19 mm2 ) (p < 0.05) before treatment and decreased vs. (35.32±0.60) mm2 in control. The circulating type IV collagen levels decreased more than 1.8 times and 1.4 times (p < 0.05), bFGF levels decreased more than 2.2 times and 1.6 times (p < 0.05) in II and I groups vs. patients before treatment (P < 0.02). Conclusions: The UDCA+L-ornithine-L-aspartate combination in long-term use has antifibrotic effects with beneficial cytoprotective properties and improves electronomicroscopic, morphometrical and cytogenetic markers of the hepatocytes and hepatocytes nuclears in ALD patients.
Journal of Hepatology 2013 vol. 58 | S63–S227