Yoon Park

A pilot study of pioglitazone treatment for nonalcoholic steatohepatitis

Nonalcoholic steatohepatitis (NASH) is a common chronic liver disease for which there is no known effective therapy. A proportion of patients with NASH progress to advanced fibrosis and cirrhosis. NASH is considered one of the clinical features of the metabolic syndrome in which insulin resistance plays a central role. This prospective study evaluates the role of insulin‐sensitizing agent in treatment of NASH. Eighteen nondiabetic patients with biopsy‐proven NASH were treated with pioglitazone (30 mg daily) for 48 weeks. Tests of insulin sensitivity and body composition as well as liver biopsies were performed before and at the end of treatment. By 48 weeks, serum alanine aminotransferase values fell to normal in 72% of patients. Hepatic fat content and size as determined by magnetic resonance imaging decreased, and glucose and free fatty acid sensitivity to insulin were uniformly improved. Histological features of steatosis, cellular injury, parenchymal inflammation, Mallory bodies, and fibrosis were significantly improved from baseline (all P < 0.05). Using strict criteria, histological improvement occurred in two‐thirds of patients. Pioglitazone was well tolerated; the main side effects were weight gain (averaging 4%) and an increase in total body adiposity. In conclusion, these results indicate that treatment with an insulin‐sensitizing agent can lead to improvement in biochemical and histological features of NASH and support the role of insulin resistance in the pathogenesis of this disease. The long‐term safety and benefits of pioglitazone require further study. (HEPATOLOGY 2004;39:188–196.)

Treatment of chronic non-A, non-B hepatitis with recombinant human alpha interferon

We treated 10 patients who had chronic non-A,non-B hepatitis with recombinant human alpha interferon in varying doses (0.5 to 5 million units) daily, every other day, or three times weekly for up to 12 months. In 8 of the 10 patients, elevated serum aminotransferase levels decreased rapidly during therapy and eventually fell into the normal or nearly normal range. In two of these patients, the interferon therapy was stopped after four months, and in both cases, a prompt return of aminotransferase activities to pretreatment values occurred. Prolonged treatment was associated with a sustained improvement in aminotransferase levels; in three cases, biopsy specimens obtained after one year of therapy showed marked improvement in hepatic histology, even though low doses of alpha interferon had been used. These preliminary findings, although not adequately controlled, suggest that long-term, low-dose alpha interferon therapy may be effective in controlling the disease activity in some patients with chronic non-A,non-B hepatitis. A prospective controlled trial is now needed to assess the role of interferon therapy in this disease.

Hepatic Failure and Lactic Acidosis Due to Fialuridine (FIAU), an Investigational Nucleoside Analogue for Chronic Hepatitis B

BACKGROUND We describe severe and unexpected multisystem toxicity that occurred during a study of the antiviral nucleoside analogue fialuridine (1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-iodouracil, or FIAU) as therapy for chronic hepatitis B virus infection. METHODS Fifteen patients with chronic hepatitis B were randomly assigned to receive fialuridine at a dose of either 0.10 or 0.25 mg per kilogram of body weight per day for 24 weeks and were monitored every 1 to 2 weeks by means of a physical examination, blood tests, and testing for hepatitis B virus markers. RESULTS During the 13th week lactic acidosis and liver failure suddenly developed in one patient. The study was terminated on an emergency basis, and all treatment with fialuridine was discontinued. Seven patients were found to have severe hepatotoxicity, with progressive lactic acidosis, worsening jaundice, and deteriorating hepatic synthetic function despite the discontinuation of fialuridine. Three other patients had mild hepatotoxicity. Several patients also had pancreatitis, neuropathy, or myopathy. Of the seven patients with severe hepatotoxicity, five died and two survived after liver transplantation. Histologic analysis of liver tissue revealed marked accumulation of microvesicular and macrovesicular fat, with minimal necrosis of hepatocytes or architectural changes. Electron microscopy showed abnormal mitochondria and the accumulation of fat in hepatocytes. CONCLUSIONS In patients with chronic hepatitis B, treatment with fialuridine induced a severe toxic reaction characterized by hepatic failure, lactic acidosis, pancreatitis, neuropathy, and myopathy. This toxic reaction was probably caused by widespread mitochondrial damage and may occur infrequently with other nucleoside analogues.

Randomized, controlled trial of recombinant human α-interferon in patients with chronic hepatitis B

Forty-five patients with chronic hepatitis B were entered into a randomized controlled trial of recombinant human alpha-interferon therapy. All patients had hepatitis B surface antigen in serum for at least 1 yr and had stable serum levels of both hepatitis B virus deoxyribonucleic acid and hepatitis B e antigen. During the 4-mo period of therapy, 10 of 31 (32%) treated patients and only 1 of 14 (7%) control patients became negative for serum hepatitis B virus deoxyribonucleic acid and deoxyribonucleic acid polymerase. All 10 patients who became negative for serum hepatitis B virus deoxyribonucleic acid subsequently had a marked improvement in serum aminotransferase activities and lost hepatitis B e antigen from serum, and 9 of them had improvement in liver histology. Comparison of responders to nonresponders indicated that female sex and a high initial level of serum aspartate aminotransferase correlated best with response to interferon therapy. These findings indicate that a 4-mo course of recombinant alpha-interferon can induce a remission in disease in approximately one-third of patients with chronic hepatitis B.

Ribavirin as Therapy for Chronic Hepatitis C: A Randomized, Double-Blind, Placebo-Controlled Trial

Hepatitis C virus (HCV) infection is a frequent cause of chronic liver disease, cirrhosis, and hepatocellular carcinoma [1, 2]. Chronic hepatitis C affects an estimated 3 million persons in the United States [3] and ranks second only to alcoholic liver disease as a cause of end-stage liver disease and as a condition necessitating liver transplantation [4]. Currently, the only therapy that has proved to be beneficial in patients with chronic hepatitis C is interferon-. Although effective in eliminating HCV infection in some cases, interferon induces long-term disease remission in only 10% to 25% of patients [5, 6]. Furthermore, interferon- therapy can have intolerable side effects and is expensive. Clearly, other forms of therapy for chronic hepatitis C are needed. Ribavirin is a broad-spectrum, oral, purine nucleoside analog antiviral agent that is similar in structure to guanosine. It is widely used in an aerosol formulation to treat respiratory syncytial virus infections in children [7]. In preliminary trials, an oral preparation of ribavirin was found to improve serum aminotransferase levels in approximately one third of patients with hepatitis C [8-10]. However, a 6-month course of ribavirin therapy was associated with little change in liver histopathology and serum HCV RNA levels. We evaluated the effect of ribavirin administered for a longer period in a randomized, double-blind, placebo-controlled trial. Methods Study Sample Patients enrolled in our trial had compensated chronic hepatitis C and no evidence of other liver disease. Entry criteria included 1) persistent elevations in serum alanine aminotransferase levels that averaged more than twice the upper limit of normal on three measurements made within the previous 6 months; 2) the presence of antibody to HCV [anti-HCV] and HCV RNA in serum; and 3) chronic hepatitis determined by liver biopsy. Exclusion criteria included therapy with other agents, such as interferon, during the previous 6 months; decompensated liver disease; pregnancy or inability to practice birth control; preexisting anemia; serologic evidence of ongoing hepatitis B, hepatitis D, or HIV infection; other significant medical illness; or alcohol abuse (> 60 g/d). Study Design and Treatment After a preliminary period of screening assessments, patients were admitted to the Clinical Center of the National Institutes of Health for medical evaluation and liver biopsy. They were then randomly assigned (using random numbers derived from published tables in blocks of 6, 8, or 10) to receive either ribavirin or placebo. Ribavirin (Virazole, ICN Pharmaceuticals, Costa Mesa, California) was administered as three 200-mg capsules twice daily. Placebo was administered in capsules that were identical in appearance to and given in the same manner as the ribavirin capsules. Therapy was continued for 48 weeks. After starting therapy, patients were evaluated in an outpatient setting once a week for the first month, once every 2 weeks for the second month, then once monthly until 6 months, and bimonthly thereafter. At each visit, patients were interviewed and examined and had blood drawn for routine biochemical and hematologic tests and tests for viral markers. Patients and caregivers were blinded to treatment assignments. The blood test results were also withheld from patients and caregivers but were monitored by an investigator who had no contact with patients during the trial. Compliance was monitored by evaluating the regularity of attendance at clinic visits, by reviewing patient diaries, and by pill count. Maintenance of the blinded nature of the study was assessed by asking patients at the end of the study whether they believed they had received ribavirin or placebo. During the last 2 weeks of treatment, patients again had medical evaluations and liver biopsies, after which the treatment code was broken. Patients who had received placebo were offered treatment with ribavirin in an open trial. Patients who had received ribavirin were followed at 1- to 2-month intervals for at least 6 months. Routine blood tests included complete blood counts and biochemical tests of liver and renal function. Selected serum samples from each patient were tested for anti-HCV (HCV EIA 2.0, Abbott Laboratories, North Chicago, Illinois). They were also tested for HCV RNA by reverse transcription polymerase chain reaction (PCR) using nested primers from the 5 noncoding region of the viral genome [11] and for amount of HCV by the branched DNA (bDNA) signal amplification assay [12] (Quantiplex, Chiron, Emeryville, California). The bDNA assay has a sensitivity of 3.5 105 genome-equivalents per mL, whereas the PCR assay can detect as few as 500 HCV RNA molecules. Genotypes of HCV were determined by reverse hybridization of HCV complementary DNA amplified from serum by reverse transcription PCR [13, 14] (Inno-Lipa, Innogenetics, Brussels, Belgium). Liver biopsy specimens taken before and after therapy were evaluated as a group and read in random order by a single investigator who was blind to the treatment status of each patient. The effect of therapy was assessed by evaluating the degree of necrosis, inflammation, and fibrosis semiquantitatively using the histology activity index [15, 16]. Statistical Analysis Changes in serum aminotransferase levels during therapy were assessed by using repeated-measures analysis of variance. The two treatment groups at entry were compared for similarity using a two-sample Student t-test. In addition, patients were classified as responders, partial responders, or nonresponders on the basis of serum aminotransferase levels during therapy. A complete response was defined as having occurred if the mean of the six bimonthly values for alanine aminotransferase levels fell into the normal range (< 41 U/L [0.69 kat/L]). A partial response was defined as having occurred if the mean of the alanine aminotransferase levels was between 1 and 1.5 times the upper limit of the normal range (41 to 62 U/L [0.69 to 1.04 kat/L]) and was less than 50% of the pretreatment value. All patients who did not have a complete or a partial response were considered nonresponders. These criteria for a definition of response are different from those used in trials of interferon- for chronic hepatitis C [5, 6] but were based on previous experience with ribavirin [8] and were established before the start of the trial. The characteristics before treatment of responders, partial responders, and nonresponders were compared using the Fisher exact test on nominal data or Wilcoxon tests on ordered data. Our trial was approved by the Institutional Review Board of the National Institute of Diabetes and Digestive and Kidney Diseases, and all patients gave written, informed consent. Results Between October 1991 and August 1992, we enrolled 58 patients in the trial (39 men and 19 women, ranging in age from 28 to 66 years [mean age, 44 years]). The known duration of hepatitis ranged from 1 to 26 years (mean duration, 6.3 years). The presumed source of hepatitis was intravenous drug use in 30 patients, blood transfusion in 19 patients, and unknown in 9 patients. Seventeen (29%) patients had previously been treated with interferon-, which had not produced a lasting beneficial effect. Before treatment, HCV RNA was detectable in the serum specimens of all patients by PCR and in the serum specimens of 51 patients (88%) by bDNA assay. The HCV genotype was 1a in 24 patients, 1b in 21 patients, 2a in four patients, 2b in three patients, 3 in four patients, and 4 in one patient. The serum HCV RNA level was not sufficient to allow genotyping in one patient. Twenty-nine patients were randomly assigned to receive ribavirin treatment and 29 were assigned to receive placebo. The initial demographic characteristics and serum biochemical and serologic features of the two groups were similar in all important ways with the exception of the mean hematocrit, which was initially higher in the ribavirin-treated group (Table 1). Table 1. Patient Characteristics by Treatment Group* Therapy was continued for 48 weeks. All patients were followed for the full 48 weeks and all had repeated liver biopsy. Compliance was similar for patients receiving ribavirin and for controls. Thus, of the 13 visits scheduled during therapy for the 58 patients, only 15 (2%) were missed (1.9% for patients receiving ribavirin and 2.1% for controls). Pill counts also suggested excellent compliance, which was similar for patients receiving ribavirin (mean, 96.0%; range, 80.3% to 100%) and for controls (mean, 96.7%; range, 87.4% to 100%). At the time of the liver biopsy that was done at 1 year, patients were asked whether they believed they were receiving ribavirin or placebo. Seven (12%) insisted that they could not tell; among the 51 patients who would guess, 38 (75%) were correct (21 of 26 ribavirin-treated patients [81%] and 17 of 25 controls [68%]). The mean serum alanine aminotransferase levels decreased among the 29 ribavirin-treated patients (P = 0.0001) but not among the 29 controls (Figure 1). The decrease in alanine aminotransferase levels seen with ribavirin therapy occurred predominantly during the first 2 months, and the levels thereafter remained constant at an average of 54% below baseline levels. The mean serum alanine aminotransferase levels (the average of the 6 bimonthly values) during therapy were below baseline levels in all ribavirin-treated patients (ranging from 87% to 9%), with the exception of 1 patient in whom the mean value increased by 49%. Alanine aminotransferase levels reached the normal range and remained within it (complete response) in 6 ribavirin-treated patients (21%) and came to within 1.5 times the upper limit of the normal range (partial response) in another 4 ribavirin-treated patients (14%). No controls had either a complete or a partial response. Thus, defined biochemical responses occurred in 10 of 29 ribavirin-treated patients (35% [95% CI

A controlled trial of naloxone infusions for the pruritus of chronic cholestasis.

To test the hypothesis that opioid agonist activity contributes to the pruritus of cholestasis, a placebo-controlled single-blinded trial of naloxone, an opioid antagonist, was conducted in eight patients with primary biliary cirrhosis. After discontinuation of all conventional antipruritic medications, one or two continuous (24-hour) IV infusions of naloxone (0.2 micrograms.kg-1.min-1) and placebo solution were administered consecutively in an order that was not predetermined. Pruritus was assessed subjectively by means of four hourly recordings of a visual analogue score. In addition, objective measurements of scratching activity that were independent of gross body movements were continuously recorded using an apparatus specifically designed to measure the frequencies associated with this activity. No side effects associated with naloxone infusions were observed. Only scratching activity data obtained for the same periods of day and night during both naloxone and placebo infusions were compared. Naloxone infusions were consistently associated with a decrease in values of the scratching activity index. In addition, in 50% of the patients the infusions were associated with a decrease in visual analogue score. The mean decrease in scratching activity ranged from 29% to 96% (mean, 50%; P less than 0.001). These findings imply that increased opioid agonist activity contributes to scratching activity in cholestatic patients.

Interferon alfa for patients with clinically apparent cirrhosis due to chronic hepatitis B

BACKGROUND The prognosis of advanced cirrhosis due to chronic hepatitis B is poor, and results of therapies, including liver transplantation, have been unsatisfactory. Little is known about the effectiveness of interferon alfa in patients with cirrhosis. METHODS Between 1984 and 1991, 18 patients with clinically-apparent cirrhosis due to hepatitis B were treated with interferon alfa at the Clinical Center of the National Institutes of Health. RESULTS Six treated patients (33%) had a sustained loss of hepatitis B virus DNA and hepatitis B e antigen (if present initially) and decrease of amino-transferase levels into the normal or near normal range. In follow-up, these 6 patients resolved all symptoms of cirrhosis and are alive and fully active. In contrast, the 12 patients who did not have a sustained loss of hepatitis B virus have had evidence of progressive liver disease, 6 have died and 4 underwent hepatic transplantation. Side effects of interferon were common and included bacterial infections (n = 5) and exacerbations of disease (n = 9). CONCLUSIONS These findings indicate that interferon alfa is effective in selected patients with mildly decompensated cirrhosis due to hepatitis B.

Increased caffeine consumption is associated with reduced hepatic fibrosis

Although coffee consumption has been associated with reduced frequency of liver disease, it is unclear whether the effect is from coffee or caffeine and whether there is an effect on hepatic fibrosis specifically. This study was undertaken to use a food‐frequency instrument for dietary caffeine consumption to evaluate the relationship between caffeine intake and liver fibrosis. Patients undergoing liver biopsy completed a detailed caffeine questionnaire on three occasions over a 6‐month period. Caffeine intake was compared between patients with mild and advanced liver fibrosis (bridging fibrosis/cirrhosis). Logistic regression was used to evaluate the association between caffeine consumption and hepatic fibrosis. One hundred seventy‐seven patients (99 male, 104 white, 121 with chronic hepatitis C virus [HCV] infection) undergoing liver biopsy completed the caffeine questionnaire on up to three occasions. Results from repeated questionnaires were consistent. Daily caffeine consumption above the 75th percentile for the cohort (308 mg = approximately 2.25 cups of coffee equivalents) was associated with reduced liver fibrosis (odds ratio [OR], 0.33; 95% confidence interval [CI], 0.14‐0.80; P = 0.015) and the protective association persisted after controlling for age, sex, race, liver disease, body mass index, and alcohol intake in all patients (OR, 0.25; 95% CI, 0.09‐0.67; P = 0.006), as well as the subset with HCV infection (OR, 0.19; 95% CI, 0.05‐0.66; P = 0.009). Despite a modest trend, consumption of caffeine from sources other than coffee or of decaffeinated coffee was not associated with reduced liver fibrosis. A reliable tool for measurement of caffeine consumption demonstrated that caffeine consumption, particularly from regular coffee, above a threshold of approximately 2 coffee‐cup equivalents per day, was associated with less severe hepatic fibrosis. (HEPATOLOGY 2010;51:201–209.)

Sec6-dependent sorting of fungal extracellular exosomes and laccase of Cryptococcus neoformans

The cell wall of pathogenic fungi such as Cryptococcus neoformans, provides a formidable barrier to secrete virulence factors that produce host cell damage. To study secretion of virulence factors to the cell periphery, sec6 RNAi mutant strains of C. neoformans were tested for virulence factor expression. The studies reported here show that SEC6 RNAi mutant strains were defective in a number of virulence factors including laccase, urease as well as soluble polysaccharide and demonstrated attenuated virulence in mice. Further analysis by transmission electron microscopy detected the production of abundant extracellular exosomes in wild‐type strains containing empty plasmid, but a complete absence in the iSEC6 strain. In addition, a green fluorescent protein–laccase fusion protein demonstrated aberrant localization within cytoplasmic vesicles in iSEC6 strains. In contrast, iSEC6 strains retained normal growth at 37°C, as well as substantially normal capsule formation, phospholipase activity and total secreted protein. These results provide the first molecular evidence for the existence of fungal exosomes and associate these vesicles with the virulence of C. neoformans.

Deregulation of FoxM1b leads to tumour metastasis.

The forkhead box M1b (FoxM1b) transcription factor is over‐expressed in human cancers, and its expression often correlates with poor prognosis. Previously, using conditional knockout strains, we showed that FoxM1b is essential for hepatocellular carcinoma (HCC) development. However, over‐expression of FoxM1b had only marginal effects on HCC progression. Here we investigated the effect of FoxM1b expression in the absence of its inhibitor Arf. We show that transgenic expression of FoxM1b in an Arf‐null background drives hepatic fibrosis and metastasis of HCC. We identify novel mechanisms of FoxM1b that are involved in epithelial–mesenchymal transition, cell motility, invasion and a pre‐metastatic niche formation. FoxM1b activates the Akt‐Snail1 pathway and stimulates expression of Stathmin, lysyl oxidase, lysyl oxidase like‐2 and several other genes involved in metastasis. Furthermore, we show that an Arf‐derived peptide, which inhibits FoxM1b, impedes metastasis of the FoxM1b‐expressing HCC cells. The observations indicate that FoxM1b is a potent activator of tumour metastasis and that the Arf‐mediated inhibition of FoxM1b is a critical mechanism for suppression of tumour metastasis.