Jay H. Hoofnagle

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.)

Pioglitazone, Vitamin E, or Placebo for Nonalcoholic Steatohepatitis

BACKGROUND Nonalcoholic steatohepatitis is a common liver disease that can progress to cirrhosis. Currently, there is no established treatment for this disease. METHODS We randomly assigned 247 adults with nonalcoholic steatohepatitis and without diabetes to receive pioglitazone at a dose of 30 mg daily (80 subjects), vitamin E at a dose of 800 IU daily (84 subjects), or placebo (83 subjects), for 96 weeks. The primary outcome was an improvement in histologic features of nonalcoholic steatohepatitis, as assessed with the use of a composite of standardized scores for steatosis, lobular inflammation, hepatocellular ballooning, and fibrosis. Given the two planned primary comparisons, P values of less than 0.025 were considered to indicate statistical significance. RESULTS Vitamin E therapy, as compared with placebo, was associated with a significantly higher rate of improvement in nonalcoholic steatohepatitis (43% vs. 19%, P=0.001), but the difference in the rate of improvement with pioglitazone as compared with placebo was not significant (34% and 19%, respectively; P=0.04). Serum alanine and aspartate aminotransferase levels were reduced with vitamin E and with pioglitazone, as compared with placebo (P<0.001 for both comparisons), and both agents were associated with reductions in hepatic steatosis (P=0.005 for vitamin E and P<0.001 for pioglitazone) and lobular inflammation (P=0.02 for vitamin E and P=0.004 for pioglitazone) but not with improvement in fibrosis scores (P=0.24 for vitamin E and P=0.12 for pioglitazone). Subjects who received pioglitazone gained more weight than did those who received vitamin E or placebo; the rates of other side effects were similar among the three groups. CONCLUSIONS Vitamin E was superior to placebo for the treatment of nonalcoholic steatohepatitis in adults without diabetes. There was no benefit of pioglitazone over placebo for the primary outcome; however, significant benefits of pioglitazone were observed for some of the secondary outcomes. (ClinicalTrials.gov number, NCT00063622.)

Recombinant interferon alfa therapy for chronic hepatitis C. A randomized, double-blind, placebo-controlled trial.

Infection with the hepatitis C virus may result in chronic liver disease for which no effective therapy is now available. We studied the effects of recombinant human interferon alfa in a prospective, randomized, double-blind, placebo-controlled trial in patients with well-documented chronic hepatitis C. Forty-one patients were enrolled in the trial, 37 of whom were later found to have antibody to hepatitis C virus. Twenty-one patients received interferon alfa (2 million units) subcutaneously three times weekly for six months, and 20 received placebo. The mean serum aminotransferase levels and the histologic features of the liver improved significantly in the patients treated with interferon but not in the patients given placebo. Ten patients treated with interferon (48 percent) had a complete response, defined as a decline in mean serum aminotransferase levels to the normal range during therapy; three others had a decrease in mean aminotransferase levels of more than 50 percent. After treatment ended, however, serum aminotransferases usually returned to pretreatment levels; 6 to 12 months after the discontinuation of interferon therapy, only two patients (10 percent) still had normal values. We conclude that interferon alfa therapy is beneficial in reducing disease activity in chronic hepatitis C; however, the beneficial responses are often transient.

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.

Mechanism of action of interferon and ribavirin in treatment of hepatitis C.

Since the identification of the hepatitis C virus, great strides have been made in the development of an antiviral therapy. As a crucial mediator of the innate antiviral immune response, interferon-α (IFN-α) was a natural choice for treatment. Whereas treatment with IFN-α alone achieved only modest success, the addition of the broad-spectrum antiviral agent ribavirin greatly improved responses. However, half of the infected individuals with chronic disease do not achieve sustained clearance of hepatitis C virus. To optimize current therapeutic strategies and to develop new therapies, a better understanding of the mechanism of action of IFN and ribavirin will be essential.

Pathogenesis, Natural History, Treatment, and Prevention of Hepatitis C

Dr. T. Jake Liang (Liver Diseases Section, National Institute of Diabetes and Digestive and Kidney Diseases [NIDDK], National Institutes of Health [NIH], Bethesda, Maryland): The identification of hepatitis C virus (HCV) as the cause of non-A, non-B hepatitis represents a technical tour de force of modern molecular medicine (1). Characterization of the viral genome and the structures and functions of viral gene products has led to a better understanding of the viral life cycle and the pathogenesis of HCV-associated disease. This knowledge will contribute to the development of an effective vaccine and better therapies. Hepatitis C virus is a member of the Flaviviridae family, which includes the flaviviruses and pestiviruses (2). There are at least 6 HCV genotypes and more than 50 subtypes. The virion contains a positive single-stranded RNA genome of 9.5 kilobases (Figure 1). The genome consists of 5 and 3 untranslated regions (5UTR and 3XR) that have little sequence variation among all genotypes and are important for translation of viral proteins and replication of the virus. The viral genome encodes a large single polyprotein of about 3000 amino acids; the N-terminal one third harbors the structural proteins, and the C-terminal two thirds contains the nonstructural proteins. The HCV structural proteins comprise the core protein and the two envelope glycoproteins E1 and E2. The nonstructural proteins, including proteases (NS2/3 and NS3), helicase (an enzyme that unwinds double-stranded nucleic acid) (NS3), and RNA-dependent RNA polymerase (NS5B), perform various functions essential for the viral life cycle (Table 1). Cleavage of structural proteins from the polyprotein is catalyzed by a host signal peptidase, whereas polyprotein cleavage in the nonstructural region requires HCV-encoded proteases. Figure 1. Schematic diagram of the hepatitis C virus genome. 5UTR 3XR Table 1. Functions of Genetic Elements of Hepatitis C Virus Hepatitis C virus enters a susceptible host either directly, through needle inoculation or transfusion of contaminated blood products, or inadvertently, through breakage of a percutaneous barrier (as exemplified by sexual or perinatal transmission) (3). The virus then enters hepatocytes or other susceptible cells, probably through a unique surface molecule or molecules, as the viral receptor (4). After uptake, the virus uncoats and releases the genome to begin replication. The viral genome first serves as the template for translation of the polyprotein. The processed nonstructural proteins then form a complex with the genome and initiate synthesis of the negative strand, which in turn functions as the template for positive strand synthesis. The replication complex probably resides in a membranous compartment in the cytoplasm, presumably derived from the endoplasmic reticulum. The RNA replicative intermediate matures and interacts with the core and envelope proteins to assemble into a virion. Although most of the replicative processes have not been defined, some nonstructural proteins clearly play critical roles in viral replication and productive infection; these proteins are therefore the focus of antiviral development. Immunopathogenesis of Hepatitis C Dr. Barbara Rehermann (Liver Diseases Section, NIDDK, NIH): Unlike other hepatitis viruses, the hepatitis C virus is more likely to cause clinically inapparent, chronic infection in persons who are otherwise considered immunocompetent. Thus, the virus is capable of circumventing an efficient immune response of the host. Components of Antiviral Immune Response The mechanisms whereby HCV circumvents immune response and establishes persistent infection are currently undefined. It is well known that the specific immune response to any viral infection is primed by macrophages and dendritic cells that present viral proteins to B cells, helper T cells, and cytotoxic T cells (Figure 2). In many viral infections, B cells produce antibodies that can clear circulating virus and protect from reinfection. For example, antibodies against the hepatitis B virus surface antigen are critical for viral clearance. Through specific T-cell receptors on the cell surface, helper T cells recognize viral peptides that are derived from phagocytosed and proteolytically cleaved HCV proteins and are presented in the context of class II MHC molecules. Figure 2. Components of the antiviral immune response. HCV On activation of their specific T-cell receptors, HCV-specific helper T cells assist with activation and differentiation of B cells as well as induction (5) and stimulation of virus-specific cytotoxic T cells. Most of these effects are mediated by different sets of immunoregulatory Th1 (interferon- and interleukin-2) or Th2 (interleukin-4, interleukin-5, and interleukin-10) cytokines. In the context of class I MHC molecules, CD8-positive cytotoxic T cells recognize HCV peptides that are synthesized and processed in infected cells (Figure 2). This encounter can lead to lysis of virus-infected cells. Together with helper T cells, cytotoxic T lymphocytes may also secrete cytokines, such as interferon- and tumor necrosis factor-, that inhibit replication and gene expression of several viruses, such as hepatitis B virus, cytomegalovirus, and rotavirus (6-8). Humoral Immune Response Hepatitis C virus can establish persistent infection despite an active humoral and cellular immune response that is generally targeted against all viral proteins. The virus may escape from the humoral immune response if the kinetics of infection and viral replication do not allow complete neutralization of the virus by HCV-specific antibodies after primary infection. Although virus-specific antibodies may interfere with viral entry into host cells and opsonize the virus for elimination by macrophages, they cannot eliminate HCV from infected cells. In addition, HCV has a high mutation rate, especially in the hypervariable region of the envelope proteins that can be recognized by neutralizing antibodies (antibodies that can bind and eliminate virus) (9, 10). Several studies have demonstrated that the humoral immune response can select HCV variants with sequence changes that allow escape from antibody recognition (11-14). However, recent studies in chimpanzees have suggested that HCV can cause persistent infection in the absence of mutations in the hypervariable region (15, 16). Thus, progression to persistent HCV infection is most likely a multifactorial process that depends on multiple aspects of virus-host interaction. Cellular Immune Response The cellular immune response probably plays an important role in the outcome of HCV infection because of its ability to recognize and eliminate virus from infected cells. Most studies have concentrated on the antigen-specific immune response that is mediated by CD4-positive helper T cells and CD8-positive cytotoxic T cells. Because chronic rather than acute infection is diagnosed in most patients, immunologic studies have been performed on patients with persistent infection who could not clear HCV. Only a few studies have analyzed the cellular immune response during the acute phase of infection. These studies suggest that the strength and quality of both helper T-cell (17, 18) and cytotoxic T-cell responses (19) differ between patients who recover and those who develop chronic infection. More important, the viral sequences that are recognized most frequently and vigorously by HCV-specific T cells vary little among all the HCV genotypes. Furthermore, several of these frequently recognized viral peptides bind with high affinity to many different class II MHC molecules, suggesting that they can be efficiently presented and recognized by patients with different MHC haplotypes (17, 20). Thus, these viral sequences could be explored for development of preventive or therapeutic vaccines against HCV. The cellular response against HCV could be interfered with in several ways. First, HCV elicits only a weak T-cell response in patients who develop chronic infection (17, 21). In the blood of patients with chronic hepatitis C, the frequency of cytotoxic T-cell precursors that are specific for individual HCV peptides is much lower than the frequency of T cells that recognize an influenza virus peptide as a recall antigen (22) or peptides of other viruses that can be cleared, such as cytomegalovirus (23). The reasons for this relative weakness of the cellular immune response are not known. Certainly, general immune tolerance or immunosuppression is not the cause of persistent HCV infection, because most chronically infected patients display normal immune responses against other viral agents (22). The emergence of viral mutants or quasi-species with sequence variations in T-cell epitopes may contribute to the apparent ineffectiveness of cell-mediated immune response (24-26). There is also increasing evidence that several HCV proteins, such as core (27), E2 (28), and NS5A (29), interfere with the immune response. Furthermore, infected hepatocytes, which lack co-stimulatory molecules, may be relatively inefficient in priming the immune system, and the liver has been proposed as the major site where activated T cells are destroyed (30). Finally, the cellular immune response is a double-edged sword. An immune response that is ineffective in clearing HCV infection may be more harmful to the liver, causing chronic inflammation, hepatocellular injury, and, over several decades, liver fibrosis and cirrhosis. Progression to persistent infection and the immunologic mechanisms of liver injury are the consequence of complicated interactions between the virus and host. Identification of immunologic correlates of viral clearance may contribute to the development of an effective vaccine and better therapy for HCV infection. Natural History of Hepatitis C Dr. Leonard B. Seeff (Division of Digestive Diseases and Nutrition, NIDDK, NIH): The natural history of hepatitis C continues to be

Routes of infection, viremia, and liver disease in blood donors found to have hepatitis C virus infection

BACKGROUND For many people infected with the hepatitis C virus (HCV), the route of exposure, risk of transmission, and severity of associated liver disease are unknown. We studied these variables in people who donated blood voluntarily. METHODS Blood donors who tested positive for HCV antibodies on enzyme immunoassay were classified according to whether the results of a confirmatory second-generation recombinant immunoblot assay (RIBA) for HCV were positive, negative, or indeterminate. The evaluations also included an assessment of risk factors, a physical examination, serial determinations of alanine aminotransferase levels and HCV serologic assays, a polymerase-chain-reaction assay for HCV RNA, testing of sexual contacts and family members, and liver biopsies in some participants who were HCV-positive by RIBA. RESULTS A total of 481 donors were studied, among whom 248 were positive for HCV by RIBA, 102 had indeterminate results, and 131 were HCV-negative. In a logistic-regression analysis, significant risk factors for HCV infection among the HCV-positive participants were a history of blood transfusion in 66 (27 percent; P < 0.001 for the comparison with RIBA-negative donors), intranasal cocaine use in 169 (68 percent, P < 0.001), intravenous drug use in 103 (42 percent, P = 0.001), sexual promiscuity in 132 (53 percent, P = 0.002), and ear piercing among men (P < 0.05). Nine of 85 sexual partners of HCV-positive donors were anti-HCV-positive; 8 had used intravenous drugs or received transfusions. HCV RNA was found in 213 HCV-positive donors (86 percent), 3 who had indeterminate results by RIBA (2 of these 3 tested positive with a more specific, third-generation RIBA), and none who were HCV-negative. Of the HCV-positive donors, 69 percent had biochemical evidence of chronic liver disease; among 77 donors positive for HCV by RIBA who underwent liver biopsy, 5 had severe chronic hepatitis or cirrhosis, 66 had mild-to-moderate chronic hepatitis, and 6 had no evidence of hepatitis. CONCLUSIONS Among volunteer blood donors, prior blood transfusion, intranasal cocaine use, intravenous drug use, sexual promiscuity, and ear piercing in men are risk factors for HCV infection. The high frequency of intravenous drug use was unexpected, because these donors had denied such use when questioned directly at the time of their blood donations.

Impaired Effector Function of Hepatitis C Virus-Specific CD8+ T Cells in Chronic Hepatitis C Virus Infection

The cellular immune response contributes to clearance of hepatitis C virus (HCV) and persists for decades after recovery from infection. The immunological basis for the inefficiency of the cellular immune response in chronically infected persons is not known. Here, we used four HLA-A2 tetramers, specific for two HCV core and two HCV NS3 epitopes, to investigate at the single-cell level effector function and phenotype of HCV-specific CD8+ T cells in 20 chronically infected and 12 long-term recovered patients. Overall, HCV-specific, tetramer+ T cells were more frequently found in PBMCs of chronically infected patients than in those of recovered patients. However, when compared with HCV-tetramer+ T cells of recovered patients, they displayed an impaired proliferative capacity. As a result of the impaired proliferative capacity, HCV-specific T cell lines derived from chronically infected patients displayed less peptide-specific cytotoxicity than those from recovered patients. In addition, proliferation and ex vivo IFN-γ production of HCV-tetramer+ cells, but not influenza-virus-specific T cells, were defective in chronically infected patients and could not be restored by in vitro stimulation with peptide and IL-2. At least three distinct phenotypes of HCV-specific CD8+ T cells were identified and associated with certain functional characteristics. In addition, impairment of proliferative, cytokine, and cytotoxic effector functions of tetramer+ T cells in viremic patients was associated with weak ex vivo HCV-specific CD4+ T cell responses. Thus, the defective functions of HCV-specific CD8+ T cells might contribute to viral persistence in chronically infected patients, and knowledge on their reversibility may facilitate the development of immunotherapeutic vaccines.

Seroconversion from Hepatitis B e Antigen to Antibody in Chronic Type B Hepatitis

Twenty-five patients with chronic type B hepatitis documented by liver biopsy were followed for 1 to 6 years with serial measurements of aminotransferase levels, hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg) and antibody (anti-HBe), and hepatitis B virus DNA polymerase. Initially, all were positive for HBsAg and HBeAg and had elevations in serum aminotransferases. In follow-up, only one lost HBsAg reactivity. In 13, however, elevated aminotransferase levels spontaneously fell to normal and have remained normal. These 13 also had a seroconversion from HBeAg to anti-HBe, and all became negative for serum DNA polymerase. Most had a fall in HBsAg titer. This seroconversion occurred concurrently with or several months before the fall in aminotransferase levels. In contrast, the 12 persons who remained HBeAg positive continued to have elevated aminotransferase levels. All 10 of these patients who were initially positive for DNA polymerase remained positive. These data suggest that many patients with chronic type B hepatitis eventually have a spontaneous remission in clinical and biochemical evidence of active disease, usually heralded or accompanied by the disappearance of HBeAg and DNA polymerase.

Effect of Vitamin E or Metformin for Treatment of Nonalcoholic Fatty Liver Disease in Children and Adolescents: The TONIC Randomized Controlled Trial

CONTEXT Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease in US children and adolescents and can present with advanced fibrosis or nonalcoholic steatohepatitis (NASH). No treatment has been established. OBJECTIVE To determine whether children with NAFLD would improve from therapeutic intervention with vitamin E or metformin. DESIGN, SETTING, AND PATIENTS Randomized, double-blind, double-dummy, placebo-controlled clinical trial conducted at 10 university clinical research centers in 173 patients (aged 8-17 years) with biopsy-confirmed NAFLD conducted between September 2005 and March 2010. Interventions Daily dosing of 800 IU of vitamin E (58 patients), 1000 mg of metformin (57 patients), or placebo (58 patients) for 96 weeks. MAIN OUTCOME MEASURES The primary outcome was sustained reduction in alanine aminotransferase (ALT) defined as 50% or less of the baseline level or 40 U/L or less at visits every 12 weeks from 48 to 96 weeks of treatment. Improvements in histological features of NAFLD and resolution of NASH were secondary outcome measures. RESULTS Sustained reduction in ALT level was similar to placebo (10/58; 17%; 95% CI, 9% to 29%) in both the vitamin E (15/58; 26%; 95% CI, 15% to 39%; P = .26) and metformin treatment groups (9/57; 16%; 95% CI, 7% to 28%; P = .83). The mean change in ALT level from baseline to 96 weeks was -35.2 U/L (95% CI, -56.9 to -13.5) with placebo vs -48.3 U/L (95% CI, -66.8 to -29.8) with vitamin E (P = .07) and -41.7 U/L (95% CI, -62.9 to -20.5) with metformin (P = .40). The mean change at 96 weeks in hepatocellular ballooning scores was 0.1 with placebo (95% CI, -0.2 to 0.3) vs -0.5 with vitamin E (95% CI, -0.8 to -0.3; P = .006) and -0.3 with metformin (95% CI, -0.6 to -0.0; P = .04); and in NAFLD activity score, -0.7 with placebo (95% CI, -1.3 to -0.2) vs -1.8 with vitamin E (95% CI, -2.4 to -1.2; P = .02) and -1.1 with metformin (95% CI, -1.7 to -0.5; P = .25). Among children with NASH, the proportion who resolved at 96 weeks was 28% with placebo (95% CI, 15% to 45%; 11/39) vs 58% with vitamin E (95% CI, 42% to 73%; 25/43; P = .006) and 41% with metformin (95% CI, 26% to 58%; 16/39; P = .23). Compared with placebo, neither therapy demonstrated significant improvements in other histological features. CONCLUSION Neither vitamin E nor metformin was superior to placebo in attaining the primary outcome of sustained reduction in ALT level in patients with pediatric NAFLD. TRIAL REGISTRATION clinicaltrials.gov Identifier: NCT00063635.