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Soluble (s)CD163 a macrophage activation marker is associated with liver disease severity in patients with Wilson’s disease
POSTER PRESENTATIONS THU-402 Soluble (s)CD163 a macrophage activation marker is associated with liver disease severity in patients with Wilson’s disease H. Groenbaek1, D.N. Gotthardt2, J. Björklund3, J. Pfeiffenberger4, H.J. Møller5, T. Bashlekova6, P. Ott1, K.-H. Weiss7. 1Department of Hepatology & Gastroenterology, Aarhus University Hospital, Aarhus, Denmark; 2Internal Medicine V, Heidelberg, Germany; 3Aarhus University Hospital, Aarhus, Denmark; 4Internal Medicine IV, University Hospital Heidelberg, Heidelberg, Germany; 5Department of Clinical Biochemistry, Aarhus University Hospital, Aarhus, Denmark; 6 Department of Internal Medicine V, University Hospital Heidelberg; 7 Internal Medicine IV, University Hospital Heidelberg, Heidelberg, Germany E-mail: [email protected] Background and Aims: Macrophage activation is present in liver biopsies from patients with Wilson’s disease, especially in advanced cases with fibrosis and cirrhosis. The macrophage activation marker sCD163 is associated with liver disease severity and portal hypertension in chronic liver diseases. We aimed to investigate macrophage activation by sCD163 levels in WD patients with and without liver disease. Methods: We investigated sCD163 levels in 151 patienst with Wilson’s Disease (68 males, 83 females) included in a WD registry and 40 patienst had cirrhosis at diagnosis. Liver disease severity was assessed by liver parameters (INR, albumin, bilirubin). Results: The median sCD163 level was 2.63 (range 0.89–24.9) and we observed higher levels in WD patients with cirrhosis (3.04 (1.22– 24.9)) compared to non-cirrhotic WD patients ((2.37 (0.89–8.02)) (P < 0.05). Further, sCD163 levels correlated positively with INR (0.84), gGT (0.52) and bilirubin (0.37); and negatively with albumin (−0.37) (P < 0.05, all). Conclusions: In conclusion, sCD163 levels are elevated in patients with Wilson’s Disease with higher levels in patienst with cirrhosis and associated with parameters reflecting liver disease severity. Thus macrophage activation may play a role in Wilson Disease liver disease progression. THU-403 Assessment of bile salt export pump (BSEP) inhibition by BSEPreactive immunoglobulins from Antibody-induced BSEP deficiency patients using a novel, cell-based assay J. Stindt1, C. Dröge1, M. Wammers1, P. Philippski1, C. Wiek2, H. Hanenberg2, D. Häussinger1, V. Keitel1. 1Department of Gastroenterology, Hepatology and Infectiology, Heinrich Heine University; 2Department of Otorhinolaryngology, Heinrich Heine University School of Medicine, Düsseldorf, Germany E-mail: [email protected] Background and Aims: We previously studied several patients presenting with recurrent intrahepatic cholestasis after liver transplantation for severe BSEP deficiency (PFIC-2). This phenotypic disease recurrence was caused by BSEP-reactive and -inhibitory antibodies formed against the allo-antigen BSEP and thus termed Antibody-Induced BSEP Deficiency (AIBD). Here we describe a novel, cell-based assay to directly measure BSEP inhibition with serum from AIBD patients. Methods: We generated stable human embryonic kidney (HEK) 293 cell lines expressing either NTCP-mCherry, BSEP-EYFP or both by lentiviral transduction and subsequent fluorescence activated single cell sorting followed by clonal isolation in hybridoma dishes. Monoclonal cell lines were analyzed for suitable transporter expression by fluorescence microscopy of both live and fixed cells. An assay procedure was established consisting of loading the cells via NTCP with [3H]-Taurocholate (TC) followed by its BSEP-mediated export into fresh medium. For assessment of BSEP inhibition by BSEPreactive antibodies, cells expressing both NTCP and BSEP were preincubated either with AIBD or control sera depleted of free bile salts or with antibodies purified from these.
Results: Direct comparison of HEK293 and their three stable derivative lines showed cellular uptake of [3H]-TC by NTCP and export by BSEP. The assay is divided into an uptake phase dominated by NTCP and an export phase dominated by BSEP. In the latter phase, re-uptake of exported [3H]-TC by the sodium symporter NTCP is prevented by exchange of sodium to choline in the extracellular medium. Using this assay, BSEP inhibition by several bile salt-free AIBD serum samples as well as purified AIBD antibodies could be shown. Conclusions: AIBD is caused by BSEP-reactive antibodies targeting extracellular parts of BSEP, effectively impairing bile salt transport into the canalicular lumen. By means of the assay presented here, extracellular inhibition by BSEP-reactive antibodies can be measured both quickly and robustly. This new diagnostic information may aid subsequent treatment of these patients as well as improve our understanding of AIBD disease progression. THU-404 Macrophage activation in patients with wilsons disease – associations with metabolic liver function, liver disease severity and fibrosis J.A.E. Björklund1, T.L. Laursen1, H.J. Møller2, P. Ott1, H. Grønbæk1. 1 Department of Hepatology and Gastroenterology; 2Department of Clinical Biochemistry, Aarhus University Hospital, Aarhus, Denmark E-mail: [email protected] Background and Aims: Wilson’s disease (WD) is a genetic disorder with abnormal copper metabolism affecting liver- and mental functions due to copper accumulation. With liver disease progression macrophage hyperplasia and liver fibrosis are prominent. Macrophage activation can be assessed by levels of the soluble (s) CD163 and the soluble mannose receptor (sMR). We aimed to investigate correlations between sCD163/sMR and liver function, liver fibrosis, and mental functions in the Danish national WD cohort. Methods: We included 29 Danish WD patients. Galactose elimination capacity test (GEC), Fibroscan, and liver ultrasound were performed for metabolic liver function, liver stiffness, and signs of cirrhosis. Neurological function was examined by the continuous reaction time (CRT) and the portosystemic encephalopathy (PSE) tests. sCD163 and sMR were quantified using in-house ELISAs. Results: The 29 patients (m/f 14/15) had a median age of 35 years (IQR 24–50). The median sCD163 level was 2.96 mg/l (1.97–3.93) and the sMR level was 0.25 (0.17–0.30). The GEC median was 1.98 mmol/ min (1.75–2.32), corresponding to 75% of expected liver function. sCD163 correlated significantly to GEC values (r = −0.49, p = 0.02), while sMR values did not (r = 0.07, p = 0.74). The median fibroscan value was 6.7 kPa (5.30–9.45), but the values did not correlate with sCD163 (r = 0.14, p = 0.46) or sMR (r = 0.22, p = 0.24). Ultrasound data suggest a normal liver in 7 patients, inhomogeneous liver in 6 patients, steatosis in 9 patients, fibrosis in one patient and cirrhosis in 3 patients. The median sCD163 and sMR levels did not differ between patients with and without cirrhosis (3.4 mg/l (3.33–5.07) vs. 2.83 (1.64–3.85), p = 0.21) and 0.30 (0.30–0.39) vs. 0.24 (0.17–0.30), p = 0.07). Regarding neurological function, we found a CRT-index median of 1.86 (1.60–2.53) and a PSE-score median of 0 (−1.50–1.00). There was no correlation between sCD163/sMR and CRT (r = −0.12, p = 0.5430)/(r = −0.19, p = 0.3184) or PSE (r = 0.07, p = 0.7199)/(r = −0.11, p = 0.5038). Conclusions: Macrophage activation in WD, assessed by sCD163, correlated significantly to the metabolic liver function (GEC) but we did not observed higher levels in patients with liver cirrhosis on ultrasound. The levels of sCD163 may contribute to the evaluation of liver function in patients with WD.
Journal of Hepatology 2017 vol. 66 | S95–S332
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Results: Direct comparison of HEK293 and their three stable derivative lines showed cellular uptake of [3H]-TC by NTCP and export by BSEP. The assay is divided into an uptake phase dominated by NTCP and an export phase dominated by BSEP. In the latter phase, re-uptake of exported [3H]-TC by the sodium symporter NTCP is prevented by exchange of sodium to choline in the extracellular medium. Using this assay, BSEP inhibition by several bile salt-free AIBD serum samples as well as purified AIBD antibodies could be shown. Conclusions: AIBD is caused by BSEP-reactive antibodies targeting extracellular parts of BSEP, effectively impairing bile salt transport into the canalicular lumen. By means of the assay presented here, extracellular inhibition by BSEP-reactive antibodies can be measured both quickly and robustly. This new diagnostic information may aid subsequent treatment of these patients as well as improve our understanding of AIBD disease progression. THU-404 Macrophage activation in patients with wilsons disease – associations with metabolic liver function, liver disease severity and fibrosis J.A.E. Björklund1, T.L. Laursen1, H.J. Møller2, P. Ott1, H. Grønbæk1. 1 Department of Hepatology and Gastroenterology; 2Department of Clinical Biochemistry, Aarhus University Hospital, Aarhus, Denmark E-mail: [email protected] Background and Aims: Wilson’s disease (WD) is a genetic disorder with abnormal copper metabolism affecting liver- and mental functions due to copper accumulation. With liver disease progression macrophage hyperplasia and liver fibrosis are prominent. Macrophage activation can be assessed by levels of the soluble (s) CD163 and the soluble mannose receptor (sMR). We aimed to investigate correlations between sCD163/sMR and liver function, liver fibrosis, and mental functions in the Danish national WD cohort. Methods: We included 29 Danish WD patients. Galactose elimination capacity test (GEC), Fibroscan, and liver ultrasound were performed for metabolic liver function, liver stiffness, and signs of cirrhosis. Neurological function was examined by the continuous reaction time (CRT) and the portosystemic encephalopathy (PSE) tests. sCD163 and sMR were quantified using in-house ELISAs. Results: The 29 patients (m/f 14/15) had a median age of 35 years (IQR 24–50). The median sCD163 level was 2.96 mg/l (1.97–3.93) and the sMR level was 0.25 (0.17–0.30). The GEC median was 1.98 mmol/ min (1.75–2.32), corresponding to 75% of expected liver function. sCD163 correlated significantly to GEC values (r = −0.49, p = 0.02), while sMR values did not (r = 0.07, p = 0.74). The median fibroscan value was 6.7 kPa (5.30–9.45), but the values did not correlate with sCD163 (r = 0.14, p = 0.46) or sMR (r = 0.22, p = 0.24). Ultrasound data suggest a normal liver in 7 patients, inhomogeneous liver in 6 patients, steatosis in 9 patients, fibrosis in one patient and cirrhosis in 3 patients. The median sCD163 and sMR levels did not differ between patients with and without cirrhosis (3.4 mg/l (3.33–5.07) vs. 2.83 (1.64–3.85), p = 0.21) and 0.30 (0.30–0.39) vs. 0.24 (0.17–0.30), p = 0.07). Regarding neurological function, we found a CRT-index median of 1.86 (1.60–2.53) and a PSE-score median of 0 (−1.50–1.00). There was no correlation between sCD163/sMR and CRT (r = −0.12, p = 0.5430)/(r = −0.19, p = 0.3184) or PSE (r = 0.07, p = 0.7199)/(r = −0.11, p = 0.5038). Conclusions: Macrophage activation in WD, assessed by sCD163, correlated significantly to the metabolic liver function (GEC) but we did not observed higher levels in patients with liver cirrhosis on ultrasound. The levels of sCD163 may contribute to the evaluation of liver function in patients with WD.
Journal of Hepatology 2017 vol. 66 | S95–S332
S175