Argyro Voumvouraki

Increased serum activin-A differentiates alcoholic from cirrhosis of other aetiologies.

Eur J Clin Invest 2012

Activin-A causes Hepatic stellate cell activation via the induction of TNFα and TGFβ in Kupffer cells

BACKGROUND & AIMS TGFβ superfamily member Activin-A is a multifunctional hormone/cytokine expressed in multiple tissues and cells, where it regulates cellular differentiation, proliferation, inflammation and tissue architecture. High activin-A levels have been reported in alcoholic cirrhosis and non-alcoholic steatohepatitis (NASH). Our aim was to identify the cell types involved in the fibrotic processes induced by activin-A in liver and verify the liver diseases that this molecule can be found increased. METHODS We studied the effect of activin-A on mouse primary Kupffer cells (KCs) and Hepatic Stellate cells (HSCs) and the levels of activin-A and its inhibitor follistatin in the serum of patients from a large panel of liver diseases. RESULTS Activin-A is expressed by mouse hepatocytes, HSCs and Liver Sinusoid Endothelial cells but not KCs. Each cell type expresses different activin receptor combinations. HSCs are unresponsive to activin-A due to downregulation/desensitization of type-II activin receptors, while KCs respond by increasing the expression/production of TNFα και TGFβ1. In the presence of KCs or conditioned medium from activin-A treated KCs, HSCs switch to a profibrogenic phenotype, including increased collagen and αSMA expression and migratory capacity. Incubation of activin-A treated KC conditioned medium with antibodies against TNFα and TGFβ1 partially blocks its capacity to activate HSCs. Only patients with alcoholic liver diseases and NASH cirrhosis have significantly higher activin-A levels and activin-A/follistatin ratio. CONCLUSIONS Activin-A may induce fibrosis in NASH and alcoholic cirrhosis via activation of KCs to express pro-inflammatory molecules that promote HSC-dependent fibrogenesis and could be a target for future anti-fibrotic therapies.

Presence of disease specific autoantibodies against liver sinusoidal cells in primary biliary cirrhosis.

AIM To investigate the presence of autoantibodies directed against liver sinusoidal cells in primary biliary cirrhosis (PBC). METHODS Liver biopsies from 21 PBC patients were studied and compared with 12 liver biopsies from disease controls [3 patients with hepatitis B (HBV) virus, 3 patients with hepatitis C virus (HCV), 3 patients with non-alcoholic steatohepatitis and 3 patients with acute alcoholic hepatitis (AAH)]. As healthy controls, we used tissue specimens adjacent to metastatic liver adenocarcinoma. Normal serum was taken from staff members of the unit. The determination of the cell type targeted by autoantibodies present in the patients sera was performed by indirect immunofluorescence (IIF) analysis using paraffin-embedded liver sections as a substrate. Sera from homologous or heterologous PBC patients or sera from the disease control group were used as primary antibodies. The presence of autoantibodies was identified using confocal microscopy. RESULTS In total, 18/21 (85.7%) PBC patients exhibited positive staining in the sinusoidal cells, 10/21 (47.6%) in lymphocytes, 8/21 (38%) in cholangiocytes and 7/21 (33.3%) in hepatocytes, when homologous serum and fluorescein isothiocyanate-conjugated immunoglobulin type G (IgG) secondary antibody were used. PBC sections incubated with heterologous PBC serum showed reduced staining (20% for sinusoidal cells, 20% for lymphocytes, 20% for cholangiocytes and 13.3% for hepatocytes). When IgM immunoglobulin, instead of IgG, was used as secondary antibody, positive staining was observed in 75% of lymphocytes, 62.5% of cholangiocytes, 37.5% of hepatocytes and 50% of the sinusoidal cells with a much stronger staining intensity. No staining was observed when either normal or PBC sera were used as a primary antibody on liver sections from the disease control group. When PBC sera were incubated with healthy control sections, weak positive staining of cholangiocytes was observed in 3/21 (14.3%) PBC serum samples. Steatohepatitis serum on PBC sections gave a positive staining of some hepatocytes and lymphocytes but no staining on viral hepatitis sections. Incubation with HBV sera stained some hepatocytes, cholangiocytes and intra-sinusoidal or portal lymphocytes of PBC, HBV and AAH patients but not HCV patients. CONCLUSION In this study, for the first time in diseased liver tissue, we have demonstrated that a large proportion of PBC patients have disease specific autoantibodies against liver sinusoidal cells.

Long-term change in incidence and risk factors of cirrhosis and hepatocellular carcinoma in Crete, Greece: a 25-year study

Background No sequential long-term data exist for Greece on the etiological evolution and incidence of cirrhosis and hepatocellular carcinoma. Therefore, we studied their etiological evolution over a period of 25 years in the island of Crete. Methods We studied 812 cases of cirrhosis (561 male, median age 69 years) and 321 cases of hepatocellular carcinoma (234 male, median age 70 years) from the database of our Center. Cases were classified into five-year periods according to incidence and etiology (hepatitis B, hepatitis C, alcohol, alcohol plus viral, and non-alcoholic fatty liver disease). Results Overall, there was an increase in the incidence of hepatocellular carcinoma. A significant fourfold reduction in the incidence of hepatitis C-related cirrhosis was observed, which was degraded from first to third place as a risk factor for cirrhosis. Alcohol gradually became the first risk factor in cirrhosis (1990-94: 36.1%, 2010-14: 52.3%) and carcinoma, while the steepest increase in incidence of cirrhosis and carcinoma was associated with non-alcoholic fatty liver disease. Conclusions The incidence of cirrhosis remained constant over the years, but the incidence of hepatocellular carcinoma increased during the last decade. Risk factors for cirrhosis and hepatocellular carcinoma have changed over the past 25 years in Crete. The initial high hepatitis C virus association has significantly decreased, with alcohol now ranking first among risk factors. Non-alcoholic fatty liver disease is continually increasing and is a prominent risk factor for cirrhosis and hepatocellular carcinoma.

Biomarkers for primary biliary cholangitis: current perspectives

Primary biliary cholangitis (PBC) is a chronic progressive cholestatic disease characterized by destruction of small- and medium-sized intrahepatic bile ducts. It is no longer a rare disease, since many new asymptomatic cases are incidentally identified. Liver biopsy is diagnostically critical but not always feasible or practical to be performed. Many potential, noninvasive, markers have been proposed to replace liver biopsy and further provide the assessment of disease severity and ultimate prognosis. In this review, we evaluated serum biomarkers proposed for diagnosis, extent of fibrosis, disease prognosis and attempts for early prediction of treatment response. Older biochemical and immunological markers are presented along with recent reports including the role of microRNAs and promising results based on proteomics and metabolomics.

Angiodrastic Chemokines in Colorectal Cancer: Clinicopathological Correlations

Aim To study the expression of angiodrastic chemokines in colorectal tumors and correlate findings with clinicopathological parameters and survival. Methods The proangiogenic factor VEGF, the angiogenic chemokines CXCL8 and CXCL6, and the angiostatic chemokine CXCL4 were measured by ELISA in tumor and normal tissue of 35 stage II and III patients and correlated with the histopathology markers Ki67, p53, p21, bcl2, EGFR, and MLH1 and 5-year survival. The Wilcoxon and chi-square tests were used for statistical comparisons. Results There was a significant increase of CXCL6 (p = 0.005) and VEGF (p = 0.003) in cancerous tissue compared to normal. Patients with lower levels of CXCL8 and CXCL4 lived significantly longer. Patients with loss of EGFR expression had higher levels of CXCL8 while p21 loss was associated with higher levels of CXCL6. Chemokine levels were not correlated with TNM or Dukes classification. Strong expression of p53 was accompanied by decreased survival. Conclusions (1) The angiogenic factors CXCL6 and VEGF are increased in colorectal cancer tissue with no association with the clinical stage of the disease or survival. (2) However, increased levels of tissue CXCL8 and CXCL4 are associated with poor survival. (3) Strong expression of p53 is found in patients with poor survival.

PWE-132 Association Between Smoking And Liver Fibrosis In Primary Biliary Cirrhosis

Introduction Conflicting data for the role that cigarette smoking may play in Primary Biliary Cirrhosis (PBC) have been reported. Some studies have suggested an association of smoking with a more advanced fibrotic stage. The aim of the present study therefore was to assess the association between smoking and a) the severity of histological findings at the time of diagnosis, b) the immunological features of a genetically homogeneous and geographically defined population of PBC patients. Methods Smoking history data were collected from 171 PBC patients of Cretan origin (163 female) using a standardised questionnaire. Diagnosis was based on standard biochemical, Immunological and histological criteria. Liver biopsy was performed in 148 patients at diagnosis. Liver fibrosis and histological inflammatory activity were semi-quantified according to a METAVIR-based classification system. Odds ratios (OR) were assessed using logistic regression analysis. Results Smoking history prior to diagnosis was reported in 56 patients (32,7%%). Twenty-six patients (15,2%) were active smokers at diagnosis. Male gender (AOR 8.19, 95% CI: 3.014–11.937), alcohol intake >20 g/d (AOR, 2.20, 95% CI: 1.029–4.099), severe steatosis (AOR, 5.31, 95% CI: 2.019–9.919)), and F3–F4 fibrosis stage (AOR 1.21 95% CI: 1.015–3.031), but not piecemeal necrosis grade, bile duct paucity and cholangitis, or immunological laboratory data, were associated with smoking history. Multiple logistic regression analysis identified smoking intensity, years of passive smoking and significant necroinflammatory histological activity as independent risk factors of advanced liver fibrosis (F3–F4 stage) at diagnosis, adjusted for age, gender, BMI and alcohol consumption. For every pack-year increase in smoking intensity there was a 3.2 times higher likelihood of advanced fibrosis (95% CI: 2.018–6.294). Conclusion Our study results confirm the previously reported link between smoking history and the risk of advanced liver fibrosis at diagnosis in PBC. The mechanism by which smoking may accelerate the histological progression of PBC is unknown and larger studies are needed to define it. Disclosure of Interest None Declared.

Reply to the Letter by Dr L. Filik

Sir, we appreciate the comments of Dr Filik on our recent study on activin A. We fully agree that activins are multifunctional proteins whose exact role is still unclear. We also agree that iron deposition in the liver definitely influence the progression of fibrosis. This is possibly due to the iron-induced production of reactive oxygen species through a Fenton reaction and subsequent activation of liver stellate cells. However, whether this influences activin A levels is unknown. In our series when ferritin levels were taken into account, there was no substantial change in the results. However, ferritin is not the ideal marker of liver iron deposition, as ferritin is also an acute phase reactant influenced by inflammation or the presence of certain malignancies [1]. A direct iron measurement in liver biopsies could offer a better insight into the possible relation of iron and activin A. We have measured by atomic absorption spectrometry liver iron in a variety of chronic liver disease (Xinopoulos and Kouroumalis, manuscript in preparation). Iron was indeed increased in alcoholic cirrhosis irrespective of the iron content of alcoholic beverages consumed, but it was also increased in alcoholic fatty liver and interestingly in chronic viral disease where activin A in our study was not increased. However, this is an indirect evidence and we agree that a more direct approach is required.

Increased serum activin a differentiates alcoholic from cirrhosis of other aetiologies

Introduction, background and aim Activin A is a molecule of the TGF superfamily, implicated in liver fibrosis, regeneration and stem cell differentiation. However, data on activins in liver diseases are very few. The authors therefore studied serum levels of Activin A in chronic liver diseases. To identify the origin of Activin A, levels in the hepatic vein were estimated and expression of Activin A was studied in isolated rat Kupffer and stellate cells. Methods 162 patients participated in the study: 39 with Hepatocellular Carcinoma (HCC, 19 viral cirrhosis associated, 20 alcoholic cirrhosis associated), 18 with Chronic hepatitis C (CHC), 47 with Primary Biliary Cirrhosis (PBC, 26 stage I–II and 21 stage IV), 22 with Alcoholic cirrhosis (hepatic vein blood available in 16), 20 with HCV cirrhosis (hepatic vein blood available in 18) and 16 patients with alcoholic fatty liver with mild to moderate fibrosis but no cirrhosis. 19 normal controls were also included. A commercially available ELISA was used for serum determinations and a semiquantitative PCR for Activin A expression in isolated rat Kupffer and Stellate cells. Results Activin-A levels were significantly increased (p<0.001) in serum of patients with alcoholic cirrhosis (median 673 pg/ml, range 449–3279), compared to either controls (149 pg/ml 91–193) or patients with viral cirrhosis (189 pg/ml, 81–480), CHC (142 pg/ml, 65–559) PBC stage I–II (100 pg/ml 59–597) and PBC stage IV (104 pg/ml, 81–579). Only patients with alcoholic cirrhosis associated HCC had significantly increased levels of activin-A (2403 pg/ml, 1561–7220 pg/ml). Activin A mRNA was strongly expressed in both Kupffer and stellate cells. Conclusions Serum levels of Activin A are increased in patients with alcohol related cirrhosis or HCC and can discriminate these patients from cirrhotics of other aetiologies.