Antiviral and Mutagenic Effects of APOBEC Deaminases A3C, A3D, and A3H in a Hepatitis B Virus Model.

Most cases of hepatocellular carcinoma (HCC) are associated with cirrhosis related to chronic hepatitis B virus (HBV) or hepatitis C virus (HCV) infection. Changes in the time trends of HCC and most variations in its age-, sex-, and race-specific rates among different regions are likely to be related to differences in hepatitis viruses that are most prevalent in a population, the timing of their spread, and the ages of the individuals the viruses infect. Environmental, host genetic, and viral factors can affect the risk of HCC in individuals with HBV or HCV infection. This review summarizes the risk factors for HCC among HBV- or HCV-infected individuals, based on findings from epidemiologic studies and meta-analyses, as well as determinants of patient outcome and the HCC disease burden, globally and in the United States.

Natural history of chronic hepatitis B in Euro-Mediterranean and African Countries

Time-dependent events in natural history of occult hepatitis B virus infection: the importance of population-based long-term follow-up study with repeated measurements

Therapeutic vaccines and immune-based therapies for the treatment of chronic hepatitis B: Perspectives and challenges

The role of hepatitis B virus integrations in the pathogenesis of human hepatocellular carcinoma

To study Hepatitis B virus (HBV) infection and its association with hepatocellular carcinoma (HCC) at the miRNA level. Three cellular models were used to investigate miRNA expression changes during HBV infection: human HepG2 hepatoblastoma cell line as a model without HBV infection; HepG2 cell line transfected with a 1.3-fold full-length HBV genome as an acute infection model; and HepG2.2.15 cell line, which is derived from HepG2 and stably transfected with a complete HBV genome, as a chronic infection model. The miRNA levels were examined using microarray technology. To explore the relationship between HBV infection and HCC genesis at the miRNA level, we downloaded from national center for biotechnology information Gene Expression Omnibus an miRNA expression dataset derived from HCC patients, most of whom are HBV carriers. We compared the miRNA expression alterations during HBV infection with those in HCC patients, by analyzing miRNA expression change profiles statistically. Seventy-seven and 48 miRNAs were differentially expressed during acute and chronic HBV infection, respectively. Among these miRNAs, 25 were in common, the intersection of which was significant under the hypergeometric test (P = 1.3 × 10⁻¹¹). Fourteen miRNAs were observed to change coherently in the acute and chronic infections, with one upregulated and 13 downregulated. Eleven showed inverse changes during the two phases of infection; downregulated in the acute infection and upregulated in the chronic infection. The results imply that common and specific mechanisms exist at the miRNA level during acute and chronic HBV infection. Besides, comparative analysis of the miRNA expression changes during HBV infection with those in HCC indicates that, although miRNA expression changes during HBV infection are distinct from those in HCC patients (P < 2.2 × 10⁻¹⁶), they exhibited significant correlations (P = 0.0229 for acute infection; P = 0.0084 for chronic infection). Perturbation of miRNA expression during chronic HBV infection was closer to that in HCC patients than that during acute HBV infection. This observation implies the contribution of miRNAs to HCC genesis from HBV infection. According to their patterns of differential expression in acute and chronic HBV infection, as well as in HCC, miRNAs of potential research interest could be identified, such as miR-18a/miR-18b, miR-106a, miR-221 and miR-101. For instance, the gradient expression alteration of miR-221 in the above three phases, which is downregulated in acute HBV infection, normally expressed in chronic HBV infection, and upregulated in HCC, indicates that it may be a key effector for progression of the disease. Our analysis provides insights into HBV infection and related HCC in relation to miRNAs, and reveals some candidate miRNAs for future studies.

HBeAg-negative chronic hepatitis B: From obscurity to prominence

Diagnosis, prevention and management of hepatitis B virus reactivation during anticancer therapy

Hepatitis B virus (HBV) infection, with more than 350 million carriers worldwide, remains a difficult management problem despite decades of research into potential therapies and regimens. The most effective anti-HBV drugs are interferon-α (IFN-α) and antiviral nucleoside analogues such as lamivudine. At best, however, only approximately one third of those treated respond to therapy with either of these drugs (1). The conundrum deepens when the treatment of children infected with HBV comes into question. Children exhibit a markedly different disease course than adults and consequently, the relevance of results of studies in adult patients to the treatment of children is unclear. HBV in itself is not hepatotoxic; it is the immune response to HBV that is hepatotoxic. An indicator of whether a patient is mounting a significant immune response is given by the serum levels of transaminases, released by damaged hepatocytes. The natural course of acute HBV infection in adults and children older than 6 years old can be subdivided in 3 stages. The first, known as the replicative stage, is characterised by seropositivity for HBeAg, high HBV DNA levels and high transaminase levels, the rise in transaminase levels reflecting the host immune response to the virus. This is usually followed by loss of HBeAg, development of anti-HBe antibodies, reduction of HBV DNA levels and normalisation of transaminases. The second stage is known as the inactive carrier state. The third stage, resolution of the infection, is characterised by HBsAg loss and anti-HBsAg seroconversion. Chronic HBV infection goes through an initial tolerance phase, with minimal liver damage but active viral replication. This is followed years later by an immune clearance phase, heralded by increased transaminases levels leading to anti-HBe seroconversion. Most patients then enter a phase of low viral replication (inactive HBsAg carrier state). The time lapse to achieve anti-HBe seroconversion is reportedly different according to age at infection. In perinatally infected Asian children, HBeAg-to-anti-HBe seroconversion usually occurs during the third or fourth decade of life (2–5), whereas in children infected horizontally in the Mediterranean area the infection runs a more accelerated course, with features of immune clearance even at first observation (6–8). The duration of infection before anti-HBe seroconversion is a risk factor for liver disease reactivation and virus replication, which in turn are related to the development of cirrhosis and hepatocellular carcinoma (HCC) (3). The probability of becoming a chronic HBV carrier is correlated with the age at infection and the efficiency of the immune system, and is highest in children infected within the first year of life. These children tend to become immunotolerant to the virus. Thus, more than 90% of infected infants, including the 5% who do not respond to HBV vaccination, become chronic carriers as compared with 6% to 10% if the infection occurs after the sixth year of life (2,9). Spontaneous HBeAg loss in chronically infected children occurs at an annual rate of 10% to 16% (10,11), whereas spontaneous loss of HBsAg is as low as 0.6%/year (12). The children who achieve earlier seroconversion are those with biochemical and/or histological evidence of active disease. Moreover, the annual HBsAg clearance rate is significantly higher in those children who are already anti-HBe positive (eg, low viral load), than in those with HBeAg (eg, high viral load) (1.7% vs 0.4%) (12). A study by Zacharakis et al., published in this issue of the Journal(13), has further investigated the difference in the course of chronic HBV infection in children, focusing on 3 different ethnic groups living in Thrace, Greece. Investigated were 173 paediatric HBsAg carriers for mode of infection and followed up for a mean period of 5.3 years. Children of Turkish origin had a much higher rate of vertical transmission, most likely reflecting the higher rate of HBeAg seropositivity in their mothers, than children of Greek origin, whether born in Thrace or those who immigrated from the former Soviet Union. Children of Turkish ancestry also displayed a higher rate of immunotolerance, characterised by normal or nearly normal transminase levels and low rates of spontaneous HBeAg seroconversion and HBV DNA loss during follow-up. The reason why this ethnic group is predisposed to vertical transmission remains to be elucidated and cannot be attributed to the HBV genotype because all of the patients had the Mediterranean genotype D, irrespective of ethnic origin. At follow-up, overall spontaneous seroconversion to anti-HBe was observed in 55% of the children, with an annual rate of 11%, similar to that reported in previous studies (10,11). Seroconversion to anti-HBeAg was accompanied by normalisation of transaminase levels and loss of HBV DNA in the great majority. As expected, anti-HBeAg and anti-HBsAg seroconversion rates were significantly lower in the immunotolerant children. The majority of children in this study (13) showed no signs of liver disease progression, at least by ultrasound examination, but 2 developed serious sequelae (1 cirrhosis and 1 HCC) within the observation period. It has been reported that children chronically infected with HBV are mostly asymptomatic, with normal or minimally abnormal liver function tests, but their liver histology often shows progressive inflammatory changes (14). Besides posing a serious infection risk to the community, chronically HBV-infected children, particularly males, have a high risk of progressing to cirrhosis and HCC (10,14–17). The likelihood of developing these complications is correlated with the length of time to achieve anti-HBeAg seroconversion, as mentioned above (3). In view of the relatively short length of follow-up, the progression to cirrhosis and HCC in 2 of 173 patients in Zacharakis and colleagues' series represents a high rate of serious complications. One of these 2 children developed fibrotic changes within 5 years of presentation. Of note, the child who developed HCC at the age of 18 years had lost HBsAg, indicating that anti-HBsAg seroconversion, a measure of effective control of HBV infection, does not necessarily protect a person from long-term complications. As mentioned above, the likelihood of developing HCC depends on the time span to achieve anti-HBeAg seroconversion. Similar findings are reported by Bortolotti et al. (7) in a 29-year longitudinal study of 99 Mediterranean children with chronic HBV infection. Although the overall prognosis of these horizontally infected children was good, 4 of 89 (4.5%) HBeAg positive were cirrhotic at presentation, and 2 of the 4 went on to develop HCC 9 and 16 years after anti-HBe seroconversion. In 2004, the American Association for the Study of Liver Diseases published a comprehensive set of guidelines outlining the management of chronic hepatitis B (1). For those children with raised transaminases and therefore active disease, the recommendations are as for adults. For the majority of children who do not fall into this category, however, little is said. Evidence is insufficient to allow the construction of reliable and accurate guidelines for the treatment of these particular patients. Interferon-α has been used in the treatment of HBV for the last 2 decades. As a single drug therapy, it has been shown to be effective in selected groups of patients. Thus, one third of children with raised transaminases respond to treatment (ie, become HBeAg negative), as opposed to <10% of those with normal transminases (1). It has been suggested that IFN-α simply accelerates seroconversion in patients who would have cleared HBeAg regardless of treatment (18) (ie, those already mounting an immune response to the virus as demonstrated by the raised transaminases). This does not diminish the importance of IFN-α treatment of course, because by reducing viral replication earlier rather than later, the risk of hepatic damage and carcinogenesis is likely to be reduced. Lamivudine, a nucleoside analogue that inhibits HBV replication, is a newer drug approved for use in children in whom it has shown to lead to 22% HBeAg seroconversion as compared to 13% in untreated controls (19). In adults, lamivudine also appears to be efficacious in patients with HBeAg-negative/HBV DNA–positive chronic hepatitis B. A significantly higher proportion of patients receive lamivudine (63%), experiencing loss of serum HBV DNA and normalization of transaminases after 24 weeks of treatment compared with patients receiving placebo (6%) (20). There are, however, 2 important problems associated with lamivudine treatment. First, the durability of the response is unconvincing; approximately one third of adult patients relapsed after 3 years in 1 study (21). The second, perhaps more important, drawback is the development of lamivudine-resistant strains, the most common being the one containing the YMDD motif. This mutation affects 20% to 40% of the treated patients, and its frequency depends on the duration of treatment. Lamivudine undoubtedly has a role to play in the treatment of chronic HBV infection, but the question is how best to optimise its use. A Korean study by Choe et al. (22), published in this issue of the Journal, has suggested that continuing lamivudine for at least 6 months after disappearance of HBV DNA and HBeAg gives a better rate of sustained response. In a comparison between a historical group of 19 children treated with IFN-α and 40 children treated with lamivudine for 12 months (followed by a further 6 months' treatment if they had become negative for HBeAg and HBV DNA), HBeAg seroconversion was significantly higher in the lamivudine-treated group than in the IFN-α group (65% vs 37%) 2 years after initiation of treatment. Of the 26 patients who responded to lamivudine, only 1 (4%) relapsed during the 2-year follow-up. There was no significant difference between the lamivudine and IFN-α groups in terms of loss of HBsAg. Although the lack of a control group and the use of historical controls are severe limitations to the Choe et al. study, the concept of reinforcing the antiviral response by continuing treatment with lamivudine for 6 months after disappearance of HBeAg and HBV DNA merits attention and further experimental address. Of note, a recent study shows that continuing treatment over 12 months in children who do not respond to lamivudine is associated with a high incidence (64%) of YMDD mutations, which offsets the improved rate of HBeAg loss and HBV DNA negativity (23). The second point made in the article by Choe et al. (22) is that patients under age 7 years respond better to lamivudine treatment than those older than 7. However, 12 of the 40 children treated with lamivudine in this study were IFN-α nonresponders. Although no information on their age is provided, it is likely that they were older, influencing negatively the response rate of the older age group. However, because a differential response to antiviral treatment according to age, with younger children responding better to IFN-α treatment, has been reported previously (24), the effect of age on treatment response requires further investigation. Another approach to the therapy of chronic HBV infection is the combined use of lamivudine and IFN-α. This combination has not dramatically improved the response rate in patients with active disease (eg, abnormal transminases), although it appears to reduce the emergence of YMDD-resistant mutations (25,26). In contrast to the results in children with active disease, the combination of lamivudine and IFN-α may be of benefit in children who are immunotolerant to the virus (the majority of children with chronic HBV infection worldwide). In a recent pilot study, 23 immunotolerant children with normal transaminase levels and inactive histology were treated sequentially with combination therapy (27). Lamivudine was given alone for 8 weeks to decrease the viral load, after which time IFN-α was added for 44 weeks to boost virus-specific immune responses. Of these patients, 82% became HBV DNA–negative by the end of the 12-month treatment period, although 3 months later only 5 children (22%) continued to be HBV DNA negative. All of these 5 children seroconverted to anti-HBeAg and 4 to anti-HBsAg. After a 3-year follow-up, the antiviral response was sustained. None of the 23 patients developed YMDD mutations. These results represent a considerable improvement over previous reports in immunotolerant children (28). This avenue of research deserves to be actively pursued in control trials because it challenges the widely held view that children with normal transaminases should not be treated (29). An effective treatment for chronic HBV infection has yet to be found. The virus may be tackled at different levels of its replicative cycle using old and new nucleoside/nucleotide analogues alone or in combination, and the immune response to the virus can be enhanced using newer forms of IFN, such as pegylated interferon. The combination of these 2 approaches should increase the number of patients responding to treatment. However, it is important to remember that chronic HBV infection, 1 of the principal health concerns worldwide, will be eliminated only through the implementation of universal HBV vaccination.

Epidemiology of hepatitis B in Europe and worldwide.

The incidence of hepatitis B virus (HBV) infection in dialysis populations has declined over recent decades, largely because of improvements in infection control and widespread implementation of HBV vaccination. Regardless, outbreaks of infection continue to occur in dialysis units, and prevalence rates remain unacceptably high. For a variety of reasons, dialysis patients are at increased risk of acquiring HBV. They also demonstrate different disease manifestations compared with healthy individuals and are more likely to progress to chronic carriage. This paper will review the epidemiology, modes of transmission and diagnosis of HBV in this population. Prevention and treatment will be discussed, with a specific focus on strategies to improve vaccination response, new therapeutic options and selection of patients for therapy.

worldwide are living with chronic hep-atitis B virus (HBV) infection, and an estimated 620,000 die annually from complications of HBV-related liver disease [1]. In the United States, the incidence of acute HBV infection has declined substantially since 1985 as a result of the availability of effective HBV vaccines and widespread immunization of infants and high-risk populations [2]. Nevertheless, approximately 43,000 new cases of acute HBV infection occur each year in the United States [3]. Further, although vaccination programs have successfully reduced the incidence, the

Targeting Innate Immunity: A New Step in the Development of Combination Therapy for Chronic Hepatitis B

Major risk factors for hepatocellular carcinoma (HCC) vary by region and population. Chronic infection with hepatitis B virus (HBV) and/or hepatitis C virus (HCV), heavy alcohol intake, aflatoxin exposure, haemochromatosis, nonalcoholic fatty liver disease, obesity, diabetes mellitus and tobacco smoking have been suggested to be risk factors of HCC. However, chronic infection with HBV remains the major cause of HCC in the world. More than 50% of HCC cases worldwide and 80% of HCC cases in most high-risk areas, such as the Asia‐Pacific region and Africa, are attributable to HBV infection. The relative risk of HCC among individuals infected with HBV ranges from 5 to 49 in case‐control studies and from 7 to 98 in cohort studies [1]. In HBV endemic regions, HBV infection is usually acquired perinatally or in childhood, whereas in HBV nonendemic areas, it is mostly transmitted during adolescence or adulthood. Newborns become chronic HBV carriers at a very high rate (about 90%), while immune-competent adults are generally described as developing chronic infection at a rate of 5‐10%. HBV subgenotype C2 is more prone to causing chronic infection than HBV subgenotype B2 following acute hepatitis B [2]. Up to 40% of patients with chronic HBV infection will develop the life-threatening complications of HCC or decompensated liver cirrhosis (LC). Chronic hepatitis B (CHB), LC and HCC are progressive liver diseases and consecutive stages of HBVassociated hepatocarcinogenesis, although CHB may precede the asymptomatic hepatitis B surface antigen (HBsAg) carrier (ASC) state and HCC does not have to pass through the CHB or LC stage. Hepatitis B e antigen (HBeAg) expression indicates active viral replication. HBeAg expression, high viral load and alanine aminotransferase (ALT) levels are associated with an increased risk of HCC. HBV subgenotypes B2 and C2 are endemic in most parts of Asia, while subgenotype B1 is endemic in Japan. Chronic infection with HBV C2 is frequently associated with an increased risk of LC and HCC in patients older than 50 years, whereas chronic infection with HBV B2 is associated with HCC or HCC recurrence in young, mostly non-cirrhotic, patients. In comparison with infection with HBV B2 or C2, infection with HBV B1 is associated with fulminant hepatitis B, a lower incidence of HCC and the development of HCC when older. Different HBV genotypes display their own distinct mutation pattern in the preS region and in the enhancer II (EnhII)/basic core promoter (BCP)/precore region of the HBV genome. Takahashi and colleagues identified HCC-associated HBV mutations through the comparative analysis of full-length HBV isolates (95% genotype C) from sera of 40 Japanese patients with HCC. They found that deletions and missense mutations in the preS2 region, A1762T and G1764A (often appearing simultaneously, termed A1762T/G1764A) and T1753C/A mutations in the BCP region and G1613A and C1653T mutations in the EnhII region were more frequent in HCC patients [3]. This pioneering work has aroused great interest in the field. Many studies pertaining to the association of HBV mutations with the risk of HCC have been published during the past decade. It has been found that the HBV preS deletion, A1762T/G1764A, T1753V, C1653T and T31C are each associated with a significantly increased risk of HCC [4]. Of the two major HBV

Preventing Infection-Associated Cancer: From Bench to Hillside