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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-kappaB 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-kappaB, as compared to NASH and controls (p < 0.01). Degrees of inflammation and fibrosis correlated with NF-kappaB 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-kappaB expression, and disease severity. This potential mechanistic link might provide multiple interesting targets for therapeutic intervention.

Objective: To assess the burden of liver disease in Portugal., Methods: Cross-sectional study conducted in 2001 among 773,187 patients, corresponding to 922,611 hospital admissions. Liver disease was defined by International Classification of Diseases (9th edition) codes 570-573. The cost of hospitalization was computed using the groups of homogeneous diagnosis defined by the Portuguese Ministry of Health., Results: In 2001 there were 12,371 patients admitted for liver-related disease, for a total of 16,607 hospital admissions, corresponding to 1.6% and 1.8% of all patients and hospital admissions, respectively. The mean age of the subjects was 57 +/- 16 years, with 8870 (72%) men and 3501 (28%) women. There were 2181 in-hospital deaths, corresponding to 17.6% of the patients admitted. Alcohol-related liver disease (ALD) represented 63% of hospitalizations, and non alcohol-related liver disease (NALD) represented 37%. However, among men there were 69% ALD, while in women this percentage was only 46% (P < 0.001). The median length of stay (interquartile range) for each admission was 8 (11) days in ALD and 8 (13) days in NALD, respectively. Complications were reported in 7446 admissions: upper gastrointestinal bleeding, 3436 (46.1%); ascites, 3425 (46.0%); encephalopathy, 2245 (30.2%); jaundice, 122 (1.6%). The cost of hospital admissions for liver disease was euro 59.4 million, corresponding to 2.4% of all hospital health care expenses., Conclusion: Although there are strong limitations depending mostly on the inadequacy of International Classification of Diseases (9th edition) regarding liver disease, present data indicate that this condition is still mainly associated with alcohol, representing an important burden in what concerns either hospitalization time or economic costs.

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.

Introduction: Spontaneous bacterial peritonitis is a common and severe complication in patients with cirrhosis and ascitis. Its prognosis clearly depends on its precocious clinical recognition and efficacious therapy., Aim: To optimize a treatment protocol, after auditing clinical efficacy and describe microorganisms implicated at our institution., Material and Methods: Retrospective study of clinical files of patients with hepatic cirrhosis with positive culture of ascitic fluid (AF) and/or an AF polymorphonuclear (PMN) count of more than 250/mm3, treated at our units between 1st January, 2000 and 31st December, 2001 (n = 38). Patients showed a median age of 49 years (30-76), 63% of which were male. Forty-eight percent were classified as belonging to Child-Pugh B class, and 52% to C., Results: First, considering cases with PMN > 250/mm3 (n = 29), antibiotics were given to all patients (cefotaxime and ampiciline). Fifty-two percent had hepatic encephalopathy, 42% had fever, 66% abdominal pain. In 42% a microorganism was isolated. Although 24% of fatal cases (only two related to infection), we noted a 73% clinical and laboratorial response. Five patients (72%) that died, showed renal failure by the time of death. Second, in all cases with positive culture of ascitic fluid (n = 21), 42% of which with PMN > 250/mm3 and 9 monobacterial nonneutrocytic bacterascites' cases, one only agent was found: E. coli in 36%, Streptococci (37%), Staphylococci (14%), and other (14%): Klebsiella oxytoca, n = 1; Salmonella enteritidis, n = 1; Enterococcus faecium, n = 1, Acinectobacter anitratus, n = 1. Only one of the agents, E. faecium (3%) showed in vitro sensitivity exclusively to ampiciline; all other were cefotaxime sensitivite., Conclusions: Our protocol will be modified, to treat patients with spontaneous bacterial peritonitis with cefotaxime, as monotherapy. Albumin infusion will also be added to the protocol, as, we found renal failure to be an important negative prognosis factor.

Background and Aim: Non-alcoholic steatohepatitis (NASH), the association of steatosis with an inflammatory response, is a novel liver disease of unknown pathogenesis and prognosis. Triacylglycerols and their precursors, the fatty acids, are the likely candidates to accumulate in the hepatocyte. Disturbed fatty acid metabolism can be involved in the pathogenesis of NASH but there is no information concerning its plasma fatty acid profile. The aim of this study was to evaluate plasma total (esterified plus free) and free fatty acids concentrations to assess the association of NASH with plasma fatty acid accumulation., Materials and Methods: Overnight fasting blood samples from 22 biopsy-proven NASH patients and of 6 matched age healthy controls were studied., Results: NASH patients had significantly higher concentration of total and free fatty acids than controls (P<0.05), higher total saturated and monounsaturated levels in both studied lipid fractions (P<0.05), mainly due to the increase of hexadecanoic, hexadecenoic and octadecenoic acids. Absolute polyunsaturated fatty acids (PUFA) concentrations were similar in both groups. The C20:4/C18:2 and the C18:1/C18:0 ratios as well as the peroxidability index were not significantly different., Conclusion: In overweight/obese patients NASH is associated with deranged fatty acid metabolism which may be involved in its pathogenesis and/or progression.

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.

Background/aims: Percutaneous Endoscopic Gastrostomy (PEG) has become a commonly-performed procedure, to provide enteral nutrition for patients who are unable to eat. The aims of this study were to evaluate the long term efficacy, morbidity and mortality of percutaneous endoscopic gastrostomy (PEG)., Material and Methods: We analysed 144 patients who underwent a PEG procedure. Survival curves were done with the Kaplan-Meier method. The indication was long-term enteral nutrition in patients unable to maintain adequate nutrition by mouth., Results: The procedure was successful in all but one case. Mean age was 62 (18-85) years, 89 (62%) males. Seven patients recovered from their primary disease and gastrostomy tube was removed. Mean follow-up was 7.3+/-10.8 (1--66) months. Survival rates at 30 days, 1 year and 3 years following gastrostomy were 82%, 36% and 14%, respectively. Survival curves were better in females (P<0.0001). In almost all cases, patients were fed with current home-prepared food, and were ambulatory. There were no differences in survival curves according to the nutritional status., Conclusions: There were few procedure-related complications, but a high short-term mortality, probably related with the underlying disease. The use of home-prepared food through the gastrostomy was very well tolerated, and should be encouraged., (Copyright 2002 Elsevier Science Ltd. All right reserved.)

Background/aims: Hepatic stellate cell activation has a major role in the pathogenesis of hepatic fibrosis, considered to constitute part of the healing response to a necroinflammatory stimulus. However, steatosis per se, has also been shown to induce this activation. This study evaluates if hepatic stellate cell activation is present, and how it correlates with steatosis, in nonalcoholic steatohepatitis, whose hallmark is steatosis., Methodology: Steatosis, hepatocyte damage, inflammation and fibrosis were graded from 0 to 3+, in liver biopsies from 15 well documented nonalcoholic steatohepatitis and 5 normal controls. Activated hepatic stellate cell activation were identified immunohistochemically using a monoclonal antibody raised against cytoplasmic alpha-smooth muscle actin, and semiquantitatively graded using a scoring method., Results: Nonalcoholic steatohepatitis patients showed significantly greater numbers of alpha-smooth muscle actin-reactive hepatic stellate cell than controls: hepatic stellate cell index of 3.6 +/- 1.9 versus 1.5 +/- 0.5, P < 0.05. The distribution of alpha-smooth muscle actin-reactive hepatic stellate cell was higher in the perivenular areas, than in the intermediate zone and portal area, with no significant association between steatosis and alpha-smooth muscle actin-expressing hepatic stellate cell. However, a significant association was found between portal and lobular inflammation and hepatic stellate cell index, r = 0.72, P = 0.0005 and r = 0.75, P = 0.0002, respectively., Conclusions: This study demonstrates that hepatic stellate cell activation occurs in nonalcoholic steatohepatitis, clearly correlating with portal and lobular inflammation, but not with steatosis, suggesting that the mechanisms implicated in fibrosis in nonalcoholic steatohepatitis are probably related with inflammation.

Background/aims: Hypergastrinaemia has been reported in liver cirrhosis; meanwhile, it is unclear whether it is associated with an increase in gastrin cell function. The serum gastrin concentration and the number of gastrin cells in antral biopsies were studied in patients with alcoholic liver disease., Methods: Immunocytochemical and quantification techniques were used to localize and determine the number of gastrin cells., Results: Slight non-significantly higher serum gastrin values were observed in the alcoholic liver disease patients compared with controls, but the individual variation within the groups was considerable. The frequency of gastrin cells did not differ between groups. However, the size of the gastrin cell nuclei was larger in patients with liver disease than in controls, indicating increased cellular activity., Conclusions: Alcoholic liver disease, with a disturbed liver function, influences the gastrin cells. The observed alterations may reflect the effect of alcohol and/or malnutrition, or may be secondary to the influence of liver disease on other regulatory peptides.

Background and Aims: Hepatic steatosis and nonalcoholic steatohepatitis (NASH) have been associated with obesity, non insulin-dependent diabetes mellitus and hyperlipidemia. The present study was designed in order to evaluate whether patients with steatosis/NASH presented common features with the metabolic syndrome., Methods: In 30 patients with nonalcoholic fatty liver the prevalence of hypertension and diabetes; the glucose/insulin profile, lipid profile, and serum leptin were evaluated and correlated with body composition and energy expenditure, assessed by bioimpedance spectroscopy and indirect calorimetry, respectively. Results were compared with a group of eight controls., Results: Obesity was present in 80% of patients, hypertension in 50% and non insulin dependent diabetes in 33%. Glucose metabolism was altered in 69%, with elevated insulin in 14 patients. Serum leptin, higher in women, was increased in patients: 33.9 +/- 38.9 vs 9.6 +/- 6.9 ng/ml, P< 0.05. There was a correlation between insulin and leptin, both of which correlated with body mass index, fat mass and percentage of body fat. Dyslipidaemia was found in 80% of patients: 45% presented low high density lipoproteins cholesterol, 58% high low density lipoproteins and 38% elevated very low density lipoproteins., Conclusions: There is a strong association between nonalcoholic fatty liver and features of the metabolic syndrome, suggesting a simultaneous insulin resistance and decreased sensitivity to leptin., (Copyright 1999 Harcourt Publishers Ltd.)

Background & Aims: Hepatic steatosis reflects the accumulation of triglycerides and free fatty acids in hepatocytes. Although lipids and their metabolites are potentially hepatotoxic, the absence of overt injury in fatty livers suggests that adaptive responses to lipid accumulation occur. Fatty acids induce mitochondrial uncoupling proteins (UCP) 2 and 3 in muscle and fat, providing a mechanism to dispose of excessive fatty acids. Although hepatocytes do not normally express uncoupling proteins, UCP-2 is expressed in hepatocytes of genetically obese mice with fatty livers, suggesting that lipids also induce UCP-2 in hepatocytes., Methods: To test whether lipids up-regulate hepatocyte UCP-2, cultures of rat hepatocytes were treated with lipid emulsions, linoleic or oleic acid, and UCP-2 expression was evaluated by Northern blotting and immunocytochemistry. Because increased reactive oxygen species (ROS) production may contribute to lipid-related UCP-2 induction, the DNA-binding activity of the ROS-activated transcription factor, NF-kappaB, was measured, and the effects of tert-butyl hydroperoxide (TBHP) and glutathione (GSH) on UCP-2 induction were also assessed., Results: Lipid emulsions increased the DNA-binding activity of NF-kappaB and resulted in a dose- and time-dependent induction of UCP-2 transcripts in cultured hepatocytes; after 24 hours, UCP-2 messenger RNA levels were increased 4.5-fold, and increased UCP-2 protein was shown by immunocytochemistry. Consistent with the possibility that ROS generated intracellularly during lipid metabolism participates in UCP-2 induction, addition of the cell-impermeable antioxidant GSH did not alter lipid-related induction of UCP-2. Furthermore, TBHP, which is known to increase hepatocyte mitochondrial ROS production, also increased UCP-2 messenger RNA levels., Conclusions: Lipids increase ROS and induce UCP-2 in hepatocytes. Thus, the liver may adapt to an excessive supply of lipid substrates by inducing UCP-2 to facilitate substrate disposal while constraining ROS production.

The liver is a target for bacterial lipopolysaccharide (LPS) and participates in the metabolic response to endotoxemia. Recently published evidence indicates that LPS increases the expression of mitochondrial uncoupling protein-2 (UCP-2) mRNAs in several tissues, including the liver. Because hepatocytes in the healthy liver do not express UCP-2, LPS was thought to induce UCP-2 in liver macrophages, which express UCP-2 constitutively. However, the present studies of cultured peritoneal macrophages indicate that LPS reduces steady state levels of UCP-2 mRNAs in these cells. In contrast, UCP-2 mRNAs are induced in hepatocytes isolated from LPS treated rats and transfection of these hepatocytes with UCP-2 promoter-reporter constructs demonstrates substantial increases in UCP-2 promoter activity. LPS induction of hepatocyte UCP-2 expression is virtually abolished by prior treatment of rats with neutralizing antibodies to tumor necrosis factor alpha (TNF). Futhermore, TNFalpha treatment induces UCP-2 mRNA accumulation in primary cultures of hepatocytes from healthy rats. Thus, hepatocytes are likely to be important contributors to endotoxin-related increases in liver UCP-2 via a mechanism that involves the LPS-inducible cytokine, TNFalpha., (Copyright 1998 Academic Press.)