Miguel Carneiro de Moura

Hepatocyte apoptosis, expression of death receptors, and activation of NF-κB in the liver of nonalcoholic and alcoholic steatohepatitis patients

OBJECTIVES:The increasing incidence of nonalcoholic (NASH) and alcoholic steatohepatitis (ASH), associated with lack of effective treatment, has prompted intensive studies on disease pathogenesis. Apoptosis is recognized as common in liver injury and may also contribute to tissue inflammation, fibrogenesis, and development of cirrhosis. In this study, we identified mechanisms of apoptosis induction in human steatohepatitis, and evaluated potential associations between apoptosis, liver pathology, and clinical presentation in NASH and ASH.METHODS:Hepatocyte apoptosis was evaluated by the TUNEL assay in 20 patients with NASH (all ambulatory), 40 with ASH (20 ambulatory, 20 hospitalized), and 20 controls. Liver biopsies were also graded for inflammation and fibrosis. Immunohistochemistry was performed for death receptors (Fas and TNF-R1), activated caspase-3, NF-κB p65, antiapoptotic Bcl-2 protein, and uncoupling protein 2 (UCP-2).RESULTS:TUNEL-positive hepatocytes were markedly increased in NASH (p < 0.05) and ASH (p < 0.01). Similar results were obtained for activated caspase-3, confirming the occurrence of apoptosis. The Fas receptor was upregulated in ASH, especially in hospitalized patients (p < 0.01), whereas TNF-R1 was expressed both in NASH and ASH (p < 0.01). In addition, patients with ASH showed a remarkable expression of active NF-κB, as compared to NASH and controls (p < 0.01). Degrees of inflammation and fibrosis correlated with NF-κB p65 expression, which in turn coincided with apoptosis albeit Bcl-2 and UCP-2 expression.CONCLUSIONS:Liver injury in NASH and ASH is associated with increased hepatocyte apoptosis mediated by death receptors. Further, apoptosis correlates with active NF-κB expression, and disease severity. This potential mechanistic link might provide multiple interesting targets for therapeutic intervention.

Delayed clearance of serum HBsAg in compensated cirrhosis B: relation to interferon alpha therapy and disease prognosis

Delayed clearance of serum HBsAg in compensated cirrhosis B: relation to interferon alpha therapy and disease prognosis

Bile acid levels are increased in the liver of patients with steatohepatitis

Background/aims The pathogenesis of steatohepatitis remains largely unknown; however, bile acids may play a role as potential mediators of liver damage. The aim of this study was to characterize bile acid profiles in liver tissue of patients with steatohepatitis. Methods Bile acid composition was determined by gas–liquid chromatography in liver tissue from patients with nonalcoholic steatohepatitis (NASH; n=15), patients with alcoholic steatohepatitis (ASH; n=14), and controls (n=8). Liver biopsies were graded for steatosis, inflammation, and fibrosis. Results Bile acids were moderately increased in liver tissue of steatohepatitis patients compared with controls (P<0.05). Deoxycholic, chenodeoxycholic, and cholic acids were elevated by 92, 64, and 43%, respectively, in patients with steatohepatitis (P<0.05). Cholic acid was the prevailing bile acid in NASH patients and in controls. More hydrophobic bile acid species were elevated in ASH patients compared with controls (P<0.05). Significant correlations were found in NASH patients between hepatic chenodeoxycholic acid and fibrosis, and between cholic acid and trihydroxy/dihydroxy bile acids and inflammation (P<0.05). In patients with ASH, cholic acid and trihydroxy/dihydroxy bile acids were correlated with steatosis (P<0.01). Conclusion This study shows a distinct pattern of bile acids in the liver of patients with steatohepatitis. Further, the association between bile acids and histological liver injury suggests an association of specific bile acids and disease progression, possibly through bile acid-induced liver injury.

Tamoxifen-associated steatohepatitis - report of three cases

The authors describe three cases of tamoxifen-associated steatohepatitis, which resulted from a daily dosage of 20 mg used as the adjuvant treatment of breast carcinoma. Liver tests became normal after discontinuation of tamoxifen.

Apoptosis and Bcl-2 expression in the livers of patients with steatohepatitis.

Objectives Apoptosis may play a role in the pathogenesis of alcoholic (ASH) and non-alcoholic steatohepatitis (NASH). In this study, we investigated the modulation of apoptosis-related liver proteins in steatohepatitis. Methods Hepatocyte apoptosis was evaluated by the TUNEL assay in liver tissue of 12 patients with NASH, 12 with ASH and in histologically normal controls. In addition, caspase-3 processing was evaluated by immunoblot analysis. Expression of death receptors, Bcl-2 family members, and NF-κB inhibitor (IκB) were determined by western blot. Liver biopsies were also graded for inflammation and fibrosis. Results Apoptotic hepatocytes were markedly increased in NASH (P<0.05) and ASH (P<0.001) as compared to controls. Active caspase-3 was also elevated in steatohepatitis (P<0.01), coinciding with upregulation of pro-apoptotic Bax (P<0.001). Further, production of tumour necrosis factor-receptor 1 was increased up to 4-fold (P<0.05). Degradation of IκB increased >70% in steatohepatitis (P<0.001). Notably, Bcl-2 was also strongly expressed (>100-fold; P<0.001). These data were significantly correlated with relative degrees of portal and lobular inflammation. Conclusion The results show that liver injury in NASH and ASH is associated with apoptosis and NF-κB activation. Anti-apoptotic Bcl-2 is strongly expressed, probably reflecting an adaptive response to obesity or alcohol-related stress.

Nonalcoholic steatohepatitis: A long-term follow-up study. Comparison with alcoholic hepatitis in ambulatory and hospitalized patients

A previous publication analyzed the clinicopathological characteristics of 105 patients with steatohepatitis: 32 nonalcoholic, 21 ambulatory alcoholics, and 52 hospitalized alcoholics; we now report an up to 12-year follow-up (mean 5.9 ± 4.7). Between 1988 and 1993, all patients with a histological diagnosis of steatohepatitis were included; necrosis, inflammation, Mallory bodies, and fibrosis were graded. Complete follow-up data were obtained in 78%. Survival curves were similar between nonalcoholic and ambulatory alcoholics; they were, however, better in nonalcoholic than hospitalized alcoholics (P < 0.0001), and in ambulatory relative to hospitalized (P = 0.0001) alcoholics. Nonalcoholics had a better prognosis than the combined alcoholic groups (P = 0.001). Patients with moderate to severe Mallory bodies and severe fibrosis had a significantly worse survival (P < 0.01), whereas severity of hepatocellular damage and neutrophil or mononuclear infiltration had no significant impact. In conclusion, alcoholic patients as a whole had a worse prognosis, yet the ambulatory subgroup had a prognosis similar to nonalcoholic patients.

Oxidative Stress in Chronic Hepatitis C: The Effect of Interferon Therapy and Correlation with Pathological Features

AIMS To evaluate the oxidative stress parameters before, during and after interferon treatment. PATIENTS/METHODS Twenty patients were treated with interferon a2b 5 MU, three times a week, subcutaneously, for 12 months. Liver biopsy was performed six months before treatment and at the six month follow-up. Chromosomal breakage studies were evaluated by the adjusted clastogenic score (ACS, normal value [nv] 1.1 +/- 2.4%). Plasma malondialdehyde (MDA) was measured according to the Yagi method (nv 6.6 +/- 1.4 nmol/mL) and total thiols using the Ellmans reagent (DTNB) (nv 9.8 +/- 1.3 micromol/g protein). A serum marker of fibrogenesis, the amino-terminal propeptide of Procollagen type III (PIIIP), was quantified by radioimmunoassay (nv 0.37 +/- 0.18 U/L). RESULTS Compared with reference samples, the plasma of patients before treatment showed an increase of ACS (9.2 +/- 3.2%, P<0.001); higher MDA values (12.6 +/- 2.7 nmol/mL, P<0.001) and total plasma sulfhydryl groups (t-SH) were decreased (6.3 +/- 1.1 micromol/g protein, P<0.001). During treatment and at the follow-up, a decrease in ACS was noticed in all patients (P<0.001), but without normalization; a decrease in MDA was seen, with progressive normalization until the end of the follow up only in sustained responders (P<0.003), while an increase of t-SH was seen, with progressive normalization until the end of follow up in all patients (P<0.005). A positive correlation of ACS with grading of inflammation was found (r=0.52, P<0.03) but not with fibrosis staging. In contrast, plasma MDA correlates with fibrosis staging (r=0.51, P<0.03) and with PIIIP (r=0.57, P<0.03) but without grading of inflammation. CONCLUSIONS The present study confirmed the presence of oxidative stress in chronic hepatitis C patients. Interferon promotes a long term inhibition of oxidative stress with concomitant improvement of activity and fibrosis. In the management of chronic hepatitis C, adjuvant therapy with antioxidants could be useful.

CLASTOGENIC FACTORS AS BIOMARKERS OF OXIDATIVE STRESS IN CHRONIC HEPATITIS C.

Background/Aims: Clastogenic factors (CFs) are composed of lipid peroxidation products, cytokines and other oxidants with chromosome-damaging properties. They are regularly observed after radiation exposure and in chronic inflammatory diseases, where they are supposed to be risk factors for carcinogenesis. It appeared of interest to investigate their presence in the plasma of patients with chronic hepatitis C. Methods: CFs are detected by chromosomal breakage studies. They were compared to malondialdehyde (MDA), total plasma thiols (t-SH), alanine aminotransferase (ALT), viral load and histological data. Results: CFs were increased in 19 of 20 patients, 16 had increased MDA levels and 15 had decreased t-SH levels. Mean values were significantly different from the 20 controls (p < 0.001). After the first 3 months of interferon treatment, all three markers showed significant improvement, but were not completely normalized. There was a positive correlation between CFs and necroinflammatory activity (p < 0.03), while MDA was correlated with fibrosis (p < 0.03). Viral load was correlated with necrosis and inflammation (p < 0.05). Conclusion: The presence of CFs in chronic hepatitis C confirms the occurrence of oxidative stress in this disease and could be useful in clinical trials for testing antioxidants. The CF test is a sensitive assay for the detection of oxidative stress and correlates with necroinflammatory activity.

Lack of effect of colchicine in alcoholic cirrhosis: final results of a double blind randomized trial.

Background Colchicine, an inhibitor of collagen synthesis, has been suggested as potentially beneficial in cirrhosis. Objective This long-term, randomized, double blind, placebo controlled trial was conducted in order to evaluate the efficacy of colchicine in alcoholic cirrhosis. Methods Ambulatory patients with biopsy proven alcoholic cirrhosis, presenting from 1989 to 1997, with no exclusion criteria (e.g. Child–Pugh C, bilirubin > 10 mg/dl and gastrointestinal bleeding in the previous 15 days), were randomized to receive orally, 5 days/week, 1 mg/day of colchicine or placebo. Main outcome measures Results were analysed on an intention to treat basis, for survival, incidence of complications, biochemical liver tests and safety. Results Twenty-nine patients received colchicine and 26 placebo; characteristics of both groups were similar. The median follow-up was 40.6 (1.4–126.3) months in the colchicine versus 42.4 (5.7–118.2) months in the placebo group (NS). No significant side effects were reported. During follow-up, there were no significant differences in compliance and alcohol abstinence (86% vs 85%). Overall survival was not statistically different (P = 0.38). Cumulative 3-year survival rates were 74.9% in the colchicine versus 91.4% in placebo group (NS). The annual incidence rate of complications was similar with colchicine or placebo: gastrointestinal bleeding, 1.5% vs 1.2%; ascites, 3.7% vs 3.7%; and encephalopathy, 1.0% vs 0.9%. The comparison of changes in biochemical parameters between groups did not show any significant difference. Conclusions Although well tolerated, colchicine does not appear to overcome the progression and natural history of long-established alcoholic cirrhosis.

Lymphocyte T helper‐specific reactivity in sustained responders to interferon and ribavirin with negativation (seroreversion) of anti‐hepatitis C virus

Abstract: Background: Seroreversion, negativation of anti‐hepatitis C virus previously positive, is sometimes found in some chronic hepatitis C‐sustained responders (SRs) to antiviral therapy.