key: cord-0010121-ws1ft8i7
authors: nan
title: Speakers Abstracts
date: 2004-04-06
journal: J Gastroenterol Hepatol
DOI: 10.1111/j.1440-1746.2004.abs_1.x
sha: f0a30afef1b3e804325f13174ac94bc804faa584
doc_id: 10121
cord_uid: ws1ft8i7

nan

For better understanding the role of transcatheter arterial chemoembolization (TACE) in the prevention of recurrence and in the improvement of the survival after curative operation, the anti-recurrence effects of postoperative prophylactic TACE were retrospectively evaluated in patients with surgically resected hepatocellular carcinoma (HCC). In this study, 823 patients admitted in our hospital who underwent curative liver resection for HCC were accepted in this study. All patients were with the liver function of Child-Pugh class A before operation and with the average tumor size of 7.93± 4.33cm. The diagnosis was confirmed in all patients after histological examination. The criterion of curative resection was removing of the tumor completely which was confirmed by intraoperative ultrasound and further proved by a histologically tumor-free margin and the normalization of serum AFP level after operation. All patients were randomized to treatment with postoperative prophylactic TACE (Group A, n=126), or without prophylactic TACE (Group B, n= 697). postoperatively.

The tumor recurrence was observed which suggested that the higher recurrence rate occurred within 7 months after operation, so the prophylactic TACE performed in the early of this stage (1~2 months after operation) might yield benefit in terms of anti-recurrence if the patient's liver function was well. Multivariate analysis using the Cox proportional hazards model identified that prophylactic TACE was significant and independent negative predictor of the recurrence. Cumulative recurrence curves for patients showed that the patients in group A recurred significantly fewer than did those in group B (p0.05). Our data indicated that prophylactic TACE significantly decreased the recurrence rate after hepatic resection within 2 years (p0.05) , while the long term efficacy of TACE was disappointing. The overall 1, 2, 3 and 5 year recurrent rates were 11.26%, 32.67%, 48.06% and 55.7% respectively in group A and 34.29%, 51.59%, 61.07% and 68.65% respectively in group B, showing no significant differences in the long term recurrence rates (more than 2 years, p0.05). In this series, prophylactic TACE was also found to be a significant and independent positive predictor of survival. This procedure could increase the postoperative survival within 2 years (p0.05)but the long term survival was still no differences between the two groups(p0.05). The 1, 2, 3 and 5 year survival rates were 81.48%, 70.71%, 52.29% and 42.52% in group A and 67.8%, 54.38%, 43.9% and 34.98% in group B. Furthermore, the relationship between the effects of TACE and the times of TACE having been done was analyzed by multivariate analysis. The median disease-free survival were 42, 27 and 19months in patients who accepted one, two and more times of TACE. In this study, more times of prophylactic TACE have failed to show better effectiveness in the survival prolongation.

Our study suggested that adjuvant chemoembolization might be effective in terms of prevention of two years recurrence when properly applied. The further improvement in recurrence prevention depended on the well-planed comprehensive treatments including TACE and other strategies.

Identification of and prevention against the predominant causative agent of a human common malignancy, such as hepatocellular carcinoma (HCC), should constitute the fundamental and ideal approach to control this cancer as well as its precursor disease, the more prevalent chronic hepatitis B. Such approach has also deepened studies on etiological and mechanistic aspects as well as the natural history of human hepatocarcinogenesis.

Qidong, China, is one of the hotspots known for high prevalence of HBV infection and HCC. This area has a relatively stable population and qualified cancer registry system developed on the basis of effective prospective studies on early detection and risk factor identification of HCC.

A large scale controlled clinical trial of universal immunization of newborns against HBV infection by Hep-B Vax involving 80,000 children was started since 1983 for studying its impact on the incidence of chronic hepatitis as its medium endpoint, and on the incidence of HCC and fatal cirrhosis as its final goal. The long term follow-up rate was recorded to be very high. Over 50% of the children in both cohorts were bled for tracing and comparing the prevalence of HBV infection and hepatic dysfunction. The protection rate of vaccination against HBV chronic carriage and hepatic dysfunction was shown to be around 75% when tested at 5-6 years, 10 to 12 and 18 to 20 years of age between the vaccinated and their age-matched control children who were randomized on community level. This WHO/China Demonstration Project, an international collaborative study between Cancer Institutes in China and Oxford had provided valuable input to promote the strategic approach to control hepatitis B and HCC in China. It has brought significant health benefit to the local children. The accumulating data and resources from this continuing study are valuable for ongoing and long term future studies.

The HB vaccination program will give major impact on chronic hepatitis prevalence and HCC incidence in the new generations. The presently available HBV carriers, HBsAg positive and negative, are vast in number and highly risky to develop HCC and/or severe cirrhosis. The clear definition of the absolute importance of the major causative agents in hepatocarcinogenesis and the development of approaches to control their exposure should be important. Recent studies had shown that in a consecutive series of 181 pathologically proven HCC patients in Qidong, all of them (181/181) had one or more HBV infection markers, such as HBsAg and anti-HBc or anti-HBc and HBV X gene unique sequences. The prevalence of serum HBsAg in local general population was 16% on a large scale survey with RIA. In contrast, 5.0% (6/119) of the HCC cases and 1.1% (3/287) of the sampled population were positive in serum anti-HCV. Pathological examinations showed that 100% (181/181) of the HCC cases were diagnosed of having chronic hepatitis, mostly chronic active. Therefore, presence of chronic hepatitis B (HB) was shown to be virtually ubiquitous in Qidong HCC patients, indicating its necessary role in hepatocarcinogenesis of Qidong. Similar findings in HBV infection status were also observed in HCC series in North China, but 11.8% (14/119) were co-infected with HCV. Prospective study showed that missense mutation of 249 codon of p53 gene in HCC was tightly associated with pre-exposure to aflatoxin on the background of HBV infection. Aflatoxin exposure 3.5 fold increased the risk of HCC whereas HBV infection greatly increased the sensitivity of the hepatocytes to aflatoxin. A cumulative dosage of 0.35 ug/kg body weight was shown to be able to cause HCC in HBV infected men among whom the specific molecular footprint of 249arg/ser mutation of p53 was left in the HCC DNA of all seven HCC cases followed and operated. This dosage was 700 fold lower than the carcinogenic dosage of aflatoxin to induce HCC in rhesus monkeys reported. 97 of the 181 HCC cases (54%) were shown to bear the p53 hotspot mutation, but no single case was free from HBV infection, indicating the co-factor role of aflatoxin in its range of exposure commonly detected in Qidong. The age specific 249arg/ser mutation rate of HCC in Qidong, when analyzed with the relevant HCC incidence data, could explain why Qidong HBV carriers were 3.5 times more risky in developing HCC than their Beijing counterpart. 50% or more of the increased HCC portion in Qidong HBsAg positive population came from the clonal growth of 249arg/ser mutants as a result of the interaction of HBV infection and aflatoxin exposure. The above facts strongly indicated that effective treatment of HBV infection using the rapidly developing chemo-immunotherapy as well as the advised reduction of aflatoxin intake under monitoring in high risk people might constitute the effective approaches to significantly reduce the risk of HCC in the large number of chronic HB patients, especially in areas of prevalence.

Gene therapy is defined as the introduction of foreign nucleic acids into cells or tissues with a therapeutic benefit. Based on the genetic classification of diseases as inherited monogenetic, acquired monogenetic or acquired complex genetic, gene therapy involves four concepts: gene repair or substitution for the therapy of inherited monogenetic diseases, block of gene expression or function and DNA vaccination for acquired monogenetic diseases, gene augmentation and DNA vaccination for acquired complex genetic diseases.

Inherited liver diseases. For inherited liver diseases, e.g., Crigler-Najjar-syndrome type I, hemophilia A or B gene repair as well as gene substitution have been shown to effectively correct the metabolic defect in vivo.

Viral hepatitis B and C. To treat chronic hepatitis B or C antiviral strategies are aimed at one or several steps of the viral life cycle: attachment of the virus to the cell membrane, internalization and uncoating, viral replication and gene expression, virus assembly and finally virion export. Several genetic antiviral strategies are being explored: blocking viral gene expression or function at different levels of the viral life cycle by ribozymes, small interfering RNAs (siRNAs), antisense oligonucleotides, interfering peptides or proteins (dominant negative mutants) as well as DNA vaccination.

Liver cirrhosis. While liver cirrhosis has so far been considered the irreversible result of progressive fibrous scarring and hepatocellular regeneration, in an animal model repeated intramuscular injections of DNA expressing human hepatocyte growth factor (HGF) resulted in complete resolution of fibrosis in the cirrhotic liver and improved survival of the animals. Another approach is "telomerase therapy" by telomerase gene delivery to correct telomerase dysfunction in liver cirrhosis. With a better understanding of the molecular pathogenesis of liver cirrhosis, novel genetic strategies for the treatment or prevention of liver cirrhosis will be developed.

Hepatocellular carcinoma. Based on the genetic classification of diseases described above, gene therapy of HCCs involves three concepts: gene substitution, gene augmentation, incl. tumor suppressor, suicide as well as cytokine genes, oncolytic viruses, and DNA vaccination, aimed at the modulation of the immune response to HCCs.

Current issues and perspectives. Genetically, liver diseases can be classified into three major categories: (1) monogenetic inherited diseases; (2) acquired monogenetic diseases; (3) acquired complex genetic diseases. Based on this genetic classification of diseases gene therapy involves four concepts: gene repair or substitution for the therapy of inherited monogenetic diseases, block of gene expression or function as well as DNA vaccination for acquired monogenetic diseases and gene substitution, gene augmentation and DNA vaccination for acquired complex genetic diseases.

Molecular medicine and gene therapy will increasingly become part of our patient management, complementing existing diagnostic, therapeutic and preventive strategies. Despite exciting developments in molecular therapy of liver diseases, the implementation of strategies to prevent or treat chronic liver diseases is of highest priority in clinical practice and has been shown to effectivley reduce liver-related morbidity and mortality.

Hepatocellualr carcinoma is one the most common cancer in the world and is strongly associated with chronic hepatitis B or C virus infection. Chronic viral hepatitis is assumed to induce important somatic mutations in the chromosomes of hepatocytes, which eventually lead to transform the hepatocytes to HCC. Research on this liver carcinogenesis has advanced rapidly and identified several mutated genes, including p53, beta-catenin, relevant to a certain proportions of HCC. However, many important genomic mutations remain elusive.

Genome-wide study of the HCC chromosomes discovered more than 10 loci that frequently amplify or lose. One common loss locus is between chromosome 4q21-24 in HCC. In order to map any putative tumor suppressor gene in this region, we did extensive allelic loss analysis and narrowed down the region commonly lost in HCC. To further confirm its importance, we studied any linkage of this loci to multiplex, familial HCC related to HBV infection. We recruited about 35 families with multiplex HCC with HBV and genotyped them with 50 micro-satellite markers on chromosome 4q. We analyzed the results for any linkage between the markers and HCC, and discovered a significant linkage with a set of consecutive markers. The lod score peaked to 3.1 in one marker that is within the common allelic loss locus as mapped before. The study confirmed the relevance of this region and allowed a further find-mapping of target tumor susceptibility gene.

One very unique nature of HBV-related HCC is a gender difference, with male dominant over female (3-7:1). Recently, we showed the level of testosterone and the activity of androgen receptor correlate with the risk of HCC among male HBV carriers. It suggested a role of the androgen axis in liver carcinogenesis. We therefore further investigated the property of androgen receptor gene in HCC to clarify its role.

Finally, in the development of HCC both human genomics and the viral genomics interact together to cause the diseases. As in the case of HBV-related HCC, viral replication level, as indicated by HBeAg, has been shown correlated with risk of HCC. Recent clinical studies also suggested the genotypes of HBV may influence the risk to develop HCC. To address the roles of HBV viral loads and genotypes in HCC, we conducted a case-control study with large number of male HBsAg carriers. The results indicated both increased viral loads (higher than 10,000 copies/ml) and genotype C are two independent risk factors for HCC.

Small interfering RNA (siRNA) is a double-stranded RNA (dsRNA) that directs the sequence-specific degradation of messenger RNA in mammalian cells. This offers the possibility of developing a new anti-viral therapy with high specificity. We have been using the siRNA approach to develop anti-HBV and anti-SARS drugs. Attempts have been made to inhibit HBV and coronavirus replication by specific siRNAs. We have succeeded in demonstrating an almost complete inhibition of HBV replication by stablyexpressed 21-mer short hairpin RNA (shRNA), which degraded HBV pregenomic RNA and mRNAs. Besides the conventional targets on HBV reverse-transcriptase, we also targeted other sites of pregenomic RNA, including DR elements, S, and X gene. The anti-HBV efficacy of siRNA is much more potent than that of lamivudine. Our results indicated that shRNAs can serve as efficient alternative anti-HBV agents. They can also be used in combination with chemotherapy, because they showed synergistic effects on the inhibition of HBV replication due to different mechanisms of drug actions.

There is a recent worldwide outbreak of severe acute respiratory syndrome (SARS) and a novel coronavirus has been identified as the etiologic agent. Based on the complete coronavirus genomic sequence and the functional domains, we have designed six synthetic 21-and 22-mer siRNAs targeting on the RNA-dependent RNA polymerase (replicase) region of the virus. Three of them are very active in inhibiting coronavirus infection and replication in cell culture system. Our studies provide the first evidence for the potential theapeutic use of suRNA in combating SARS.

Occupational hazards exist in any health care setting especially as they relate to the risk of acquiring infections following exposure to blood-borne pathogens. Specialists in gastroenterology often are asked to provide expert advice on the prevention and management of occupational exposures to HBV and HCV including recommendations for postexposure prophylaxis. In 1993, an estimated 1450 health-care personnel (HCP) in the United States became infected with HBV through exposure to blood and other body fluids. This represented a 90% decrease from 1985, primarily as a result of the requirements for vaccination by the Occupational Safety and Health Administration (OSHA). An estimated 100 to 200 HCP have died annually from liver failure, cirrhosis or cancer over the past 15 years because of the chronic sequelae of HBV infection acquired in the workplace decades earlier.

Transmission of HBV and HCV is highly dependent on 1) the type of exposure (e.g., needlestick vs. mucocutaneous), 2) type of fluid (e.g., blood vs. saliva), 3) infectivity of the source (viral concentration), and 4) susceptibility of the exposed. In hospital settings, the most common route of infection is following a needle stick or other sharp-instrument injury. The risk of acquiring clinical hepatitis B from blood that is HBeAg positive following a hollow needle injury is about 30% (range 22-31%). HBV concentrations in these patients often exceed 10 9 genomes/mL. Transmission rates are almost 10-fold lower (about 3% with a range from 1-6%) when the blood is HBsAg and anti-HBe positive. However, this lower risk is dependent on whether the blood contains an HBV precore mutation. Such individuals have significantly higher levels of replicating HBV and are positive for HBV DNA at levels usually exceeding 500,000 genomes/mL. Other bodily secretions and excretions (e.g., saliva) may be positive for HBsAg but contain infectious HBV that is 100 to 10,000 times less than that found in the blood. The successful experimental transmission of hepatitis B to children by the oral route using human serum containing high-titered virus, although with apparently minor clinical consequences, has been cited as supporting the possibility that oral transmission may occur under natural conditions through contaminated saliva or nasopharyngeal fluid. The subsequent detection of HBV in saliva intensified interest in saliva as a mechanism for transmitting HBV. It is postulated that saliva is contaminated by virus entering the mouth through the crevicular fluid at the gingival surface following rinsing of the mouth with water or after mastication. Alternatively, peripheral blood lymphocytes contaminating saliva may be another source of HBV DNA. In three separate animal studies, five anthropoid apes were found to be susceptible to HBsAg-positive saliva, but only by parenteral routes (subcutaneous and intravenous). No hepatitis B occurred in 13 primates that received HBV-contaminated saliva by the oral route. It is concluded that unless the inoculum is large or the concentration of virus is high, oral transmission by saliva or by objects contaminated with saliva is probably rare in nature, although percutaneous transmission via a human bite has been reported.

In contrast to hepatitis B, HCV is not as efficiently transmitted following occupational exposure to blood. The average incidence of anti-HCV seroconversion after accidental percutaneous exposure from an HCV positive source is 1.8% (range 0-7%) similar to the rate observed in HBsAg/anti-HBe positive subjects. In at least one study, only hollow-bore needle sticks resulted in transmission of HCV when compared to other routes of exposure (suture needle or sharps injury, mucous membrane contaminations, and skin contaminations). Similarly, the risk for transmission from other body fluids is expected to be neglible, in contrast to HBV.

For comparison, the occupational risk of transmission after a percutaneous exposure to HIV infected blood has been estimated to be about 0.3% (95% CI: 0.2-0.5%). Hollow-bore needles and deeper injuries appear to increase the risk of infection. As is true for HBV and HCV, the average risk after a mucous membrane exposure is approximately 0.09% (95% CI: 0.006-0.5%). The risk for transmission following exposure to other fluids has not been adequately documented but is considered to be much lower than that observed for mucocutaneous exposure.

HCP should be educated concerning the risk for and prevention of blood-borne infections, including the need to be vaccinated against hepatitis B. For preexposure immunoprophylaxis, hepatitis B vaccine is given at 0,1 and 6 months by intramuscular injection in the deltoid muscle to achieve optimal protection. Longer intervals between the last dose results in higher final titers of anti-HBs. If the vaccination schedule is interrupted after the first dose, the second dose should be given as soon as possible, with the second and third dose separated by an interval of at least 2 months. If the third dose is delayed, administration can proceed whenever convenient. To ensure that protective levels of antibody have been generated, blood from at-risk HCP should be examined by a quantitative anti-HBs assay after the basic immunization series has been completed. A report from the laboratory that the anti-HBs test is "positive" or "reactive" is not satisfactory because the level of protection may be inadequate. Based on quantitative anti-HBs results, vaccination status can be divided into four groups: (a) nonresponders, no detectable anti-HBs; (b) inadequate responder or hyporesponder, positive for anti-HBs, but with a concentration of less than 10 mIU/mL; (c) low responder, with a concentration of 10 to 100 mIU/mL; and (d) normal or adequate responder, with a concentration of greater than 100 mIU/mL. A fifth category, high responder, can be used for anti-HBs responses of greater than 1000 mIU/mL. These designations have some clinical relevance in that nonresponders or inadequate responders continue to be at risk of acquiring HBV, whereas risk of infection is low to absent in the other categories. Nonresponders or inadequate responders should be examined for evidence of HBV infection as a reason for their inability to generate an adequate anti-HBs response. In the absence of this discovery, booster doses Ú consisting of one to three additional injections of vaccine using the standard dose or four times the standard dose (e.g., the dialysis formulation) Ú are recommended. Persons who do not respond to an initial 3-dose vaccine series have a 30-50% chance of responding to a second 3-dose series. In general, the anti-HBs concentrations remain low in these individuals. Most experts will agree that low responders also should be encouraged to receive a booster inoculation 2-5 years after their last injection because breakthrough disease seems to occur earlier and more frequently in this group. In one study in homosexual men, 26% of 139 non-or hyporesponders had serologic and/or clinical evidence of HBV infection, with 44% of these subjects displaying HBsAg in the sampled specimen. Six of these subjects (4.3% of the total group) became persistently infected.

OSHA requires the use of gloves whenever performing exposure-prone procedures. Gloves worn during examinations or procedures are designed primarily to prevent cross-infection. Modern gloves consist of rubber, either natural or synthetic, and a lubricant. Natural rubber gloves are usually made from latex. The major protein component of the rubber is hevein, which is a water-soluble, thermostable and trypsin-sensitive protein. Synthetic rubber gloves are made of polyvinyl chloride and methylacrylate or polychloroprene (Neoprene). Latex gloves are useful when manual dexterity and tactility are important, when the risk of exposure to contaminants is high, and when performing tasks that might stress the glove material such as handling sharp instruments. Synthetic rubber gloves are not as flexible and do not reseal when punctured. Lubricants facilitate donning the gloves. They may be powder or liquid leading to the designation as powder or powder-free. Some powder-free gloves are coated with a nonreactive hydrogel polymer lining (Biogel) that is physically bonded to the natural rubber. This facilitates donning with wet or dry hands.

Latex rubber-associated problems include allergy to proteins in the latex or to the chemicals added during the latex manufacturing process. The former results in an antibody-mediated type I immediate hypersensitivity reaction whereas the additives cause a delayed, contact dermatitis-type reaction occurring 48-96 hours after exposure. To avoid the latter, nonlatex gloves can be worn beneath the latex gloves or low-additive gloves might be available. The frequency of latex-induced hypersensitivity ranges from 3%-11%. HCP with latex hypersensitivity may develop localized pruritus, stinging, or discomfort over the gloved hand followed in 5-20 minutes with redness, swelling, and a wheal-and-flare reaction that disappears within 1-2 hours without treatment. Conjunctivitis, generalized urticaria, rhinitis, sneezing, dyspnea, palpitations, dizziness, and laryngeal edema may ensue as can anaphylactic shock. Approximately 10% of the anaphylactic reactions that occur in patients may be due to latex allergy and may occur in association with gloved procedures (e.g., liver biopsies, endoscopies, rectal examinations). A history of atopy may be meaningful in determining potential risk. Latex-sensitive HCP should wear only nonlatex gloves, such as those made from Neoprene. Finally, airborne starch particles may transport the allergens found in the gloves and incite asthma and rhinitis.

What advice can I give our surgical and hepatology colleagues regarding the use of gloves. They should understand that glove perforation subjects the wearer to pathogens during invasive procedures whereas intact gloves are impervious to transmissible viruses. It has been estimated that a surgeon risks more than one HBV infection per lifetime (now preventable by vaccination). It has been demonstrated that 61% of the gloves worn by scrub nurses had one or more punctures compared with 24-37% of surgeons' gloves. The highest risk of glove perforations occurred among general surgeons compared to other specialties. Most of the perforations are self-inflicted and occur during closure of the wound. Major operations involving the use of a mass closure technique carry a high risk of glove puncture. Most tears are on the digits or distal interphalangeal crease of the nondominant hand. Most punctures go unrecognized. Double gloving confers some protection. In one study, the puncture rate of the inner gloves was only 2% indicating that double gloving may maintain a barrier between the patient and the surgeon in four out of five cases. A decrease in tactile sensitivity and manual dexterity are the presumed reasons that this practice is not more common. Care, experience and good surgical technique remain the only effective measures that can reduce accidental surgical injuries.

Following a potential exposure, several things should be done. The site of the wound that was in contact with blood or body fluids should be washed immediately with soap and water. Mucous membranes should be flushed with copious amounts of water. The use of antiseptics is not contraindicated, but may not be beneficial. Similarly, no evidence exists to suggest that squeezing the site of the injury to express fluid reduces the risk of transmission. An accident report should be completed with as much information as possible (date and time of exposure; details of the procedure being performed and the device being used; the type of exposure; the type and amount of fluid/tissue involved in the exposure; the infectious status of the source; and the susceptibility of the exposed person). Details about counseling, postexposure management and follow-up also are important. In general, no postexposure prophylaxis is indicated for contamination of unbroken skin or for those who inadvertently share food or utensils with a person who is infected with HBV or HCV unless there are extenuating circumstances. In the event of a human bite, both the person bitten and the person who did the biting should be evaluated. If the infection status of the source patient is known, this may be sufficient to confirm or exclude risk to the exposed person. If the infection status is not known, the source patient should be tested for HBV and HCV after obtaining an informed consent. Testing should be performed without delay by enzyme immunoassay (EIA). Tests can be modified to detect HBsAg within 2 hours. A negative anti-HCV or HBsAg result is an excellent indicator that the blood-borne pathogen is not present. The likelihood that the source person may be in the preseroconversion window period of their infection is extremely small. Confirmation of the reactive results with a supplemental test should be done to complete the testing process, but are not necessary to make initial decisions regarding postexposure management. Direct viral assays (HBV DNA and HCV RNA) are not recommended for immediate decision making purposes because of the time required to obtain a result, but might be a supplementary consideration. For those situations where the source is unknown or cannot be tested, management should be based on the likelihood of transmission such as the prevalence of disease in a population group from which the contaminated material was derived.

Management of Exposures to HBV Unvaccinated HCP exposed to an HBsAg positive source should immediately receive an intramuscular dose of hepatitis B immune globulin (HBIG) at 0.06 mL/kg along with an initial dose of HBsAg vaccine. The HBIG and vaccine should preferably be administered within 24 hours of the accident and at different sites. Their effectiveness after 7 days is unknown. If a significant delay is anticipated in obtaining or dispensing the HBIG, conventional immune globulin containing anti-HBs (0.06 mL/kg) could be substituted for the HBIG. This provides immediate (and perhaps sufficient) protection at a lower concentration of anti-HBs until the proper tests can be performed or HBIG can be located and dispensed. I favor a four-dose immunization regimen (0, 1, 2 and 6-12 months) and/or the use of the dialysis formulation concentration of HBsAg if the risk warrants such measures (e.g., deep puncture wound with a hollow-bore needle containing blood from an HBeAg positive source or a delay of several days between the injury and prophylaxis). In other situations, the conventional regimen and dosing is sufficient. For exposure to an unknown source or to an HBsAg negative source, routine hepatitis B vaccination should be initiated.

For previously vaccinated HCP, no treatment is necessary if the HBsAg source is negative regardless of the worker's anti-HBs status. For exposed persons who are known responders to the hepatitis B vaccine, no postexposure prophylaxis is necessary regardless of the HBsAg status of the exposure source. Conversely, exposed persons who are known not to have responded to the vaccine or to have had an inadequate response should receive HBIG and reinitiate the vaccine series or be given two doses of HBIG at 4-6 week intervals if the source is HBsAg positive or a known high-risk source. For those HCP who are unaware of their anti-HBs vaccine status, and who are exposed to an HBsAg positive or unknown source, their anti-HBs status can be determined by testing. If adequate levels of anti-HBs are present (>10 mIU/mL), no treatment is required. For less than an adequate anti-HBs response, a vaccine booster should be administered along with HBIG if the source is HBsAg positive. Some experts would disagree with delaying treatment in the "antibody response unknown" group exposed to an HBsAg positive source until the exposed worker's anti-HBs test results are known. For these individuals, postexposure prophylaxis should be similar to that recommended for the known nonresponder group. Should an exposed person become infected with HBV, that individual should be carefully monitored during the acute phase of the disease. Treatment with a nucleoside (lamivudine) or nucleotide (adefovir) analogue or with interferon should be withheld unless the patient's clinical condition begins to deteriorate. This is because <0.4% of hospitalized hepatitis B patients will develop fulminant hepatitis following an acute infection, and only 1-3% of immunocompetent individuals will develop chronic hepatitis B. The rest will recover completely.

The source should be tested for anti-HCV and ALT. The likelihood that the source would be in the preseroconversion window period of HCV infection in the absence of liver enzyme abnormalities is extremely small, so performing an HCV RNA test is generally not indicated. For the person exposed to an HCV-positive source, testing for anti-HCV and ALT activity should be performed at baseline and at 3 and 6 months postexposure. The baseline test is to exclude an existing infection. An earlier diagnosis can be achieved by performing an HCV RNA test at 6 and 12 weeks. Until the 12-week samples are analyzed (including the HCV RNA results), the exposed worker should be advised to practice safe sex to avoid transmitting HCV during the early stage of infection when the concentration of virus is highest and anti-HCV has not yet appeared.

Although early studies in which conventional immune globulin was administered prior to or at the time of exposure indicated some protection against the development of chronicity, this is now of academic interest only. Since 1994, all lots of conventional and intravenous immune globulin have been rendered free of anti-HCV (and HCV RNA). Regardless, guidelines are being formulated for the early treatment of exposed workers who contract overt acute hepatitis C using antiviral therapy. This is because chronic hepatitis occurs in 55-76% of these patients. Several previous studies showed a beneficial effect of treating patients with interferon alpha or beta that resulted in a significant reduction in the progression of acute to chronic hepatitis C. To resolve many of the issues raised in these early studies, a large prospective multicenter clinical trial was conducted with a more representative group of patients. Forty-four patients with acute hepatitis C were identified. The mean ALT level was 885 IU/L + 554 with the lowest level being 140 IU/L. The average time from infection to treatment was 89 days (range, 30-112) which was usually 2-4 weeks after the first signs or symptoms of disease. Treatment consisted of interferon alfa-2b at 5 million units daily for 4 weeks, then three times a week for an additional 20 weeks. After another 24 weeks of follow-up, 43 patients (98%) had undetectable levels of HCV RNA.

Occupational transmission of blood-borne viruses from HCP to patients can occur. This has been amply documented for hepatitis B primarily when the health-care worker was HBeAg positive or a precore mutation in the HBV genome existed (40 infected HCP transmitting HBV to 404 patients). For HCV, there have been 4 reports involving 4 HCP and 224 infected patients. The highest number of transmissions (217 HCV infections and 75 HBV infections) was the result of poor infection control practices.

In many situations, performance of invasive exposure-prone procedures has been implicated in transmission to patients. All breaches of the skin or epithelia by sharp instruments are by definition invasive. Exposure-prone procedures are those invasive procedures where there is a risk that injury to the worker may result in the exposure of the patient's open tissues to the blood of the worker. These include procedures where the worker's gloved hands may be in contact with sharp instruments, needle tips or sharp tissues (e.g. spicules of bone or teeth) inside a patient's open body cavity, wound or confined anatomical space where the hands or fingertips may not be completely visible at all times. Higher risk exposure-prone procedures include open cardiothoracic surgical procedures including sternal opening and closure, and major gynecological surgical procedures (e.g. caesarian section, hysterectomy). These specialties also have relatively high rates of needlestick injury of HCP that may explain the greater risk. Other specialties where this risk is increased include accident and emergency staff, dentistry (including hygienists), ENT surgical procedures, midwifery, orthodontics and paramedics. Simple endoscopic procedures have not been considered exposure prone, but should be avoided by an infected endoscopist if a significant risk of biting of the worker's fingers is deemed to be present such as in a violent or seizuring patient.

In the United Kingdom, Health Servce Guidelines recommended that HCP who are HBsAg and HBeAg positive should not undertake exposure prone procedures. A new circular (2000/20) recommended that hepatitis B infected HCP who are HBeAg negative should have their viral loads measured and those who have viral loads >1000 genome equivalents/mL should not perform exposure-prone procedures in the future. The American College of Surgeons recommends that the surgeon who is HBeAg positive should obtain expert medical advice and take appropriate measures to prevent disease transmission to patients. In 1991, the Center for Disease Control and Prevention recommended that HCP who are infected with HBV (and who are HBeAg positive) should not perform exposure-prone procedures unless they have sought counsel from an expert review panel

Ultimately, a vaccine is only as effective as the system which delivers it. Globally, about 35 million children each year remain unvaccinated, and millions more do not receive the benefits of important underutilized vaccines like hepatitis B (HB). The Global Alliance for Vaccines and Immunization (GAVI) was established in 1999 to renew immunization for the 21 st century by forging a common vision and developing new ways for partners to work together. The related Vaccine Fund has also been established with a gift of $ 750 million from the Bill and Melinda Gates Foundation, which has grown to one billion dollars with contributions from donor governments.

The Fund provides HB, Hib and YF vaccines, and auto-disable syringes and support for delivery infrastructure for children in the 75 poorest countries. Already, more than 60 of 75 eligible countries have been approved for GAVI/Vaccine Fund support, and HB vaccine has been approved in 44 additional countries. In 2002, 10 million additional children in 33 countries got GAVI supplied HB vaccines preventing > 1million carriers and ~ 100,000 future deaths.

In 1990 about 20 countries were using HB vaccine routinely for children, and by 2000 the number of countries doing so increased to 110. By the end of 2003, 151 of the 192 (79%) WHO member states will be using HB vaccine in their routine infant immunization programs. One third of the world's newborns live in China, India, and Indonesia and all three have special programs with GAVI. China will deliver HB vaccine to all newborns free of charge to parents. Indonesia will give a birth dose of HB vaccine, delivered at home, to all newborns in an innovative delivery device called UNIJECT. India has begun more than 30 demonstration projects delivering HB vaccine with the goal of routine HB immunization in their next 5-year plan. During the last 3 years great advances in the global control of hepatitis B have occurred.

Over 140 countries have already introduced universal vaccination against HBV to neonates.The safety and efficacy of such vaccines have been demonstrated unequivocally as reflected in the reduction of HBsAg carrier rates and hepatocellular carcinoma, especially in countries in Asia and in Africa. New generations of HBV vaccines containing Pre-S/S epitopes and DNA vaccines have been developed. Such vaccines may be useful in special risk groups such as non-responders to conventional HBV vaccines including older adults, obese people, healthcare workers, patients on renal dialysis, transplant patients, and patients with non-HBV chronic hepatitis. There is also a need to improve the speed of induction of immune memory against HBV in vaccinees who need immediate protection i.e. pending travel or elective surgery. The future of such vaccines depends on their immunogenicity, cost and safety profile. Pre-S/S hepatitis B vaccines produced in mammalian cells have been shown to induce faster and higher seroprotection rates against HBV, using fewer doses as compared to yeast derived vaccines.Recently, it has been suggested that vaccines against HBV may also be usefull for intervention in persistent HBV infection. Preliminary data obtained in limited clinical trials, suggest that some HBV vaccines may suppress HBV replication when administered repeatedly to patients with persistent HBV infection and hepatocellular injury. Efficacy of such an intervention, remain to be explored. Introduction of new adjuvants instead of alum may induce cellular and humoral immune responses designed to bypass immune tolerance to HBV.

According to the national seroepidemiological survey conducted in 1992~1995, the prevalence of HBsAg carriers in the general population of China was 9.7%, with the estimated number of 120X10 6 . The morbidity and mortality of chronic liver disease were 1.6/1,000 and 24.9/100,000, with the estimated number of 2x10 6 and 300x10 3 , respectively. The mortality rate of hepatocellular carcinoma (HCC) was 14.8/100,000, with the estimated number of 180x10 3 .

In order to prevent HBV infection, the Ministry of Public Health, China has implemented following strategies: 1. Screening blood donors; 2. Using disposable syringes and needles; 3. Government-led health education campaigns; 4. Universal immunization of newborn infants; 5. Immunization of preschool children and high-risk population.

In 1986 a homemade HBV vaccine derived from plasma was approved for marketing. In 1992 a homemade HBV CHO (Chinese hamster ovary cell) recombinant vaccine was approved for pilot production, and for formal production in 1996. The Merck Sharp & Dohme yeast recombinant vaccine produced by the National Vaccine and Serum Institute (NVSI) and the Shenzhen Kangtai Company (SZKT) was approved for marketing in 1996. A total of 60 million doses of the vaccine are produced per year in China at present.

The efficacy rates of HBV vaccines for interruption of mother-to-infant transmission were 86.6% for plasma derived vaccine of the National Vaccine and Serum Institute (NVSI), 80.0% for the CHO recombinant vaccine of the Changchun Vaccine and Serum Institute (CCVSI), 86.7% for the yeast recombinant vaccine of the National Vaccine and Serum Institute (NVSI), and 90.0% for the yeast recombinant vaccine of the Shenzhen Kangtai Company (SZKT), respectively.

The anti-HBs seroconversion rates of primary school children after immunization were 96.0% for the plasma derived vaccine of NVSI, 94.5% for the CHO recombinant vaccine of CCVSI, 92.5% for the yeast recombinant vaccine of NVSI, and 99.3% for the yeast recombinant vaccine of SZKT, respectively.

The Ministry of Public Health, China has integrated HBV vaccination into the EPI program administration since 1992. All newborn infants should be given three doses of HBV vaccine since 1st January 1992 (one each at birth, 1 month and 6 months). For regions without screening of pregnant women for HBsAg: 10 mg plasma derived HBV vaccine or alone to all newborn infants within 24 hrs after birth, and then 10 mg plasma derived HBV vaccine at 1 and 6 months after first dose, respectively. For regions with screening of pregnant women for HBsAg: 30 mg plasma derived HBV vaccine alone to all infants within 24 hrs after birth, then 10~30 mg plasma derived HBV vaccine at 1 and 6 months after first dose, respectively; or HBV vaccine plus HBIg to infants born to HBsAg+ mothers within 24hrs after birth, then 10~30 mg HBV vaccine at 1, and 6 months after the first dose, respectively. HBV vaccination is 10 mg HBV vaccine alone to all preschool children without HBV markers at 0-1-6 schedule for preschool children and 20 mg HBV vaccine at 0-1-6 schedule for high-risk population. The plasma derived HBV vaccine has been replaced by the yeast recombinant vaccine since 2000.

A national survey was conducted in 1999 in 31 provinces of China in order to evaluate HBV vaccine coverage in children less than 3 years of age. The survey used cluster sampling method to select children aged 18~34 months, with a total number of 25,878. The HBV vaccine coverage in infants under 12 months of age was 88.5% for urban areas and 62.7% for rural areas, respectively. HBV vaccine coverage in different provinces or autonomous regions of China was different. 11 provinces had the coverage of 7.8%~49.9%, 5 provinces 53.6%~65.3%, 9 provinces 74.4%~83.8%, and 6 provinces 86.9%~99.0%.

The adequate vaccination with 3 doses among children less than 12 months in China was 63.5%(7.8%~99.0%). The first dose vaccinated within 24 hrs after birth was 29.0% (2.1%~69.9%), and the first dose vaccinated within 48 hrs after birth was 54.0% (25.2~92.1%). 10 provinces had 7.4~19.0% of the coverage of children with adequate 3 dose HBV vaccination, 16 provinces 21.3~35.0%, 5 provinces 56.6~68.9%. 12 provinces 7.4~19.0% of infants receiving the first dose of HBV vaccine within 24hrs after birth, 14 provinces 21.3~35.0%, and 5 provinces 56.6~68.9%.

Factors associated with not receiving HBV vaccine were as follows: vaccine not provided at local hospitals (36.6%~37.7%), not aware of HBV vaccination (32.2%~34.4%), and vaccine charge is too expensive (20.4%~22.5%). The percentage of children receiving the 1st dose of HBV vaccine within 24 hrs was associated with the birthplace. It was 65.5%(207/316) for hospitals of county level, 38.6%(100/259) for hospitals of town level, and 21.5%(56/261) for homes.

The HBsAg carrier rates in vaccinated children were 1.1%, significantly lower as compared with 15.8% in the control group. The efficacy of HBV vaccine in immunized children was 93.0%, with the 95% confidence interval of 92.1%~94.0%. The efficacy of HBV vaccine in children was 44.8%~99.9% at the 9 th year after initial vaccination. The positive rates of HBsAg and anti-HBs were 1.9% and 50.9% for the vaccinated group, respectively, as compared with 16.7% and 33.3% for the control group, with the efficacy of 88.6% 15 years after initial vaccination in Longan county, Guangxi Zhuang Autonomous Region. The mortality rates of HCC per 100,000 in children aged 10~19 years at Longan county, Guangxi Zhuang Autonomous Region decreased rapidly after HBV vaccination. It was 5.7 /100000 in 1969~1988 (before vaccination), and 2.6/100000 in 1989~1992 (after vaccination) and no HCC case was found in 1993~1996.

HBV has been classified into 7 genotypes (A-G). More recently, a new genotype (H) has been reported in Central America. There is growing evidence in support of a role of HBV genotypes in the natural history of HBV infection and response to antiviral therapy. Numerous studies from Asia provide strong evidence that HBV genotype B is associated with less active and more slowly progressive liver disease compared with genotype C. This may be related to the observations that patients with HBV genotype B undergo spontaneous HBeAg seroconversion at a younger age and are more likely to have sustained remission of liver disease after seroconversion. The relation between other HBV genotypes and activity or progression of liver disease, and HBeAg seroconversion has not been properly examined. Data on the relation between HBV genotypes and HBV replication capacity are also lacking. HBV genotype is closely related to the selection of the precore stop codon mutation (G 1896 A). Genotypes B, D, and some subtypes of C are more commonly associated with the G 1896 A mutation than genotype A. HBV genotype is also related to the dual core promoter variant (A 1762 T, G 1764 A), being more commonly found in association with genotypes A and C.

Several studies reported a correlation between HBV genotypes and response to interferon therapy. One study of 64 German patients found that the rate of interferon-related HBeAg seroconversion was higher among patients with genotype A than those with genotype D. Two studies of interferon-treated patients from Taiwan and Hong Kong found that the rate of HBeAg loss was higher among patients with genotype B compared to those with genotype C. A higher rate of HBeAg loss among genotype B patients compared to genotype C patients was also observed in a study using pegylated interferon. In a recent study of pegylated interferon with or without lamivudine, sustained HBeAg loss was observed in 47%, 44%, 28%, and 25% of patients with genotypes A, B, C, and D, respectively.

The relation between HBV genotype and response to nucleoside(t)e therapy has not been well-studied. Several studies involving small numbers of patients suggest that patients with genotype B have higher rates of lamivudine-related HBeAg seroconversion, more durable response, and less resistance compared to patients with genotype C. However, these observations were refuted by others. One study found that genotype A was associated with a slightly higher rate of lamivudine resistance than genotype D.

The relation between HBV genotype and response to adefovir dipivoxil was studied in 694 patients. HBV DNA suppression at the end of 48-week treatment was comparable across the 4 major genotypes A-D. However, the number of patients with HBeAg seroconversion was too small for a definitive conclusion on the relation between HBV genotype and adefovir-related HBeAg seroconversion.

In summary, there is strong evidence for an association between HBV genotype and response to interferon therapy. Additional studies are needed to determine if there is an association between HBV genotype and response to nucleoside(t)e therapy. Future clinical trials on treatment of chronic hepatitis B should include stratification for HBV genotypes so the relation between HBV genotype and treatment response can be more accurately examined. These data may help in designing treatment algorithms according to HBV genotype as in the case of treatment of chronic hepatitis C.

Guangzhou, Nanfang Hospital, China HBV genotype B recombination Among hepatitis B virus genotypes, the B and C are most prevalent in China. To further study on the inside story of the intertypes, a study was carried out with a panel of sera collected from chronic HBV carriers living in various parts of China. The B and C and B/C recombination of 136 sequences were analyzed including 83 by restriction fragment length polymorphism and 53 by phylogenetic tree and bootscanning on full genome (23 in present study and 30 of Chinese origin from GenBank). The results showed that the genotype B in China was exclusively a subgroup of recombination with genotype C and both shared the same pre-C/C fragment. Now that the prototype B was not found in China, it was unlikely that the recombination was from coinfection of B and C. Because in pre-C/C region a number of linear epitopes and mutational hot spots distributed, it is inferred that may be a pattern of mutations. The selection pressure estimated by using a maximum likelihood approach showed that the ratio of nonsynonymousto-synonymous substitutions (d N /d S) varied more extensively in recombined pre-C/C region, providing a strong support to the mutation mechanism. Perhaps, the hybrid B has generated during HBV history and co-evolved with other genotypes over a very long period. The original B was running away under immune pressure, and the hybrid B, which seemed to possess higher evolutionary potential, was gradually becoming the exclusive B prevalent with genotype C in China.

The pre-C/C peptide is most important for T cell immune reaction and closely related with the disease activity. Since it is so, we are interesting in that whether there is still different clinical relevance between genotype B and C sharing the same core gene. HBV genotype B and C were determined in consecutive 203 HBeAg-positive patients, including 185 with chronic hepatitis and cirrhosis verified histologically and 18 with decompensated cirrhosis diagnosed clinically. The higher levels of ALT ( > 5.1•ULN) and of serum HBV DNA (>10 8 cps/ml) seemed more frequent in patients with type B (54.1% and 55.0%, respectively), but were of no significance (both p > 0.05). The distribution of genotype B and C in patients with various liver necroinflammatory diseases was not significantly different (p = 0.760), and the fibrosis results was also somewhat similar, as P = 0.536 in various severity groups between type B and C.

A prolonged and individualized interferon alpha regimen was carried out in a portion (n=166) of above patients. The response between higher and lower ALT group at the end of treatment was different (p=0.068), and between higher and lower histology activity index, when inflammatory scoring interacted by fibrosis, was significantly different (p=0.0399). The decreasing levels of serum HBV DNA were closely correlated with the increasing sustained response (p=0.0095). However, there was no statistical difference between the sustained responses of the patients with genotype B and C (p=0.2282).

Our results were somewhat different compared to the published literature Many Japanese studies showed that genotype B was more favorable than genotype C for the disease severity and outcome of the infection, while the results reported from Chinese patients were not so consistent. Some studies showed that the patients with genotype C had a more active and aggressive course, particularly had a more advanced liver disease (cirrhosis and hepatocellular carcinoma). However, it seemed to have some change in reports with time. For example, a report showed that the genotype C tended to have a higher level of serum HBV DNA and a higher frequency of advanced liver diseases, but both had no statistically The results of current study seemed to be somewhat different compared to the published literature, however, with the knowledge on recombination of genotype, the inconsistency might be explainable.

Who to treat? Current treatment of chronic hepatitis B has limited long-term efficacy. The patient's age, severity of liver disease, likelihood of response, and the possibility of adverse effects and complications should be considered before deciding on treatment.

Patients with mild chronic hepatitis should be monitored ; therapy should be considered only if there is evidence of moderate to severe activity during follow-up.

Antiviral therapy should be considered if there is active HBV replication (HBV-DNA above 10 5 copies/ml) and persistent elevation of aminotransferases. Patients with well compensated cirrhosis should be treated according to the above recommendations. Patients with decompensated cirrhosis should be treated in specialised liver units, where they can be considered for antiviral therapy and/or liver transplantation.

Recombinant interferon alpha lamivudine/adefovir dipivoxil are approved for use in many countries. No randomised controlled trials have compared all three agents. The bulk of data available refers to monotherapies, and the efficacy of suitable combination therapies is currently being evaluated. Thus a consensus document that summarises the optimal treatment of hepatitis B will require regular revision in the light of new data. Decisions about antiviral therapy should take into account the limited long-term efficacy of the three main therapeutic agents available, their side effects, costs and the predictive factors for response. Full discussion with the patient regarding the pros and cons of different strategies should lead to a joint decision about management.

The following strategies are recommended for patients with HBe-Ag positive moderate or severe chronic hepatitis without cirrhosis. A 4-6 months course of interferon alpha (5 MU daily or 9-10 MU thrice weekly, or 6 MU/m 2 thrice weekly in children) may be used as initial therapy. If interferon is contraindicated, ineffective or poorly tolerated, lamivudine or adefovir should be considered.

For patients with HBe-Ag negative moderate or severe chronic hepatitis without cirrhosis, the following strategies are recommended. A 12-24 month course of interferon alpha, 5-6 MU thrice weekly may be considered as initial therapy. If interferon is contraindicated, ineffective or poorly tolerated, lamivudine or adefovir therapy should be considered.

China bears over one-third of the total global burden of chronic hepatitis B infection, with more than 120 million people infected for life. Nationwide vaccination is effective and successful.. However, many patients chronically infected with HBV and require treatment to prevent or control serious liver damage. It has been estimated that 10 to 20 million people living in China today could benefit from effective treatment.

Fortunately, promising drug of therapies are now available and offer real hope to the millions of Chinese people with chronic hepatitis B. Therefore, the conduct of objective and valid clinical trial is the prerequisite for the approval of new therapeutic drug. Clinical trials in China should accord to the GCP( Good Clinical Practice ) principle and Helsinki Decoration to fulfill the international standard. And all clinical trials were under the supervision of Ministry of Health and State Food and Drug Administration( SFDA ) to ensure the quality of study.

In recent 10 years, we have paid special attention to the clinical trial of new anti-HBV therapeutic drugs. The trial for lamivudine has been completed and the conclusion of entecavir, adeforvir and peglated interferon alpha 2a trials will be finalized in 2004-2005. The clinical trial of lamivudine began in 1996, approved as a new drug in 1998 and manufactured domestically in 2000. Phase III and IV trials and followed up studies were completed in 2003. 429 patients and 2200 patients were enrolled in those trials respectively. A total of 429 cases with HBeAg positive chronic hepatitis B were randomized 3:1 in a double-blind , controlled trial to receive either lamivudine or placebo respectively for 12 weeks, followed open-label lamivudine treatment and followed-up for 6 years. 227 patients completed 5 years treatment. At the end of 5 years the serum HBV DNA and ALT levels continued to be suppressed in most of patients except those YMDD HBV variants were emerged. For patients with baseline ALT >2 ULN, HBeAg loss and seroconversion rates were 54% and 50% respectively by the end of 5 years. The durability of seroconversion was 82% and it was significantly correlated with baseline ALT and HBV DNA levels( bDNA assay ). .70% of cases developed YMDD variants at week 260 associated with some degree of HBV DNA rebound and flared up of ALT levels.

Lamivudine were stopped in about 20 % of patients with YMDD variants and the rest of patients still got benefit in continuing lamivudine therapy. For the safety profiles, there was no increase in the frequency or intensity of adverse events with extended lamivudine treatment and the drug is well tolerated throughout 5 years period. In the phase IV trial, 2200 both HBeAg positive and negative, and HBV DNA positive chronic hepatitis B adult and adolescent patients were enrolled. That study further verified the efficacy and safety results reported in phase III trial. In addition, liver histology and health related Quality Of Life assays were also improved. Enticavir is a novel anti-HBV agent with potent selective activity against HBV replication. The phase II trial done in China has been completed. Chronic hepatitis B patients both with HBeAg positive or negative, with HBV DNA positive were enrolled. 216 patients were randomized ( 1:1:1 ) into entecavir 0.l mg, 0.5mg daily and placebo three groups.. Entecavir 0.1mg and 0.5mg doses were superior to placebo for the primary endpoint of reducing HBV DNA level. The mean changes from baseline in HBV DNA levels at day 28 for the entecavir 0.1 mg, 0.5 mg and placebo groups as determined by bDNA assay were -2.51, -2.73, and -0.12 log 10 MEq/mL(p<0.0001).. There were no meaningful differences between entecavir and placebo groups in the incidence of adverse events on treatment. The open-label 0.5 mg study is undergoing. In addition, phase III trial and a subgroup of entecavir therapy for lamivudine failure patients will be completed at the end of 2004. Adeforvir, anti-HBV remedy has been approved in USA, European countries and Hong Kong. A phase II and III trials for chronic hepatitis B and for the patients with lamivudine failure are undergoing. Results will be revealed in 2004 and 2005. We are also involved in an international cooperation of peglated interferon alpha 2a in the treatment of chronic hepatitis B. The phase II results has be published recently and the phase IV trial will be completed soon.

Conclusion: Clinical trial of anti-HBV therapeutic remedies is progressing actively and rapidly in China. It is urgent need to train more eligible investigators, physicians, clinical pharmacologists and related team-workers to reach international standard in order improving the research quality.

The clinical significance of hepatitis B virus (HBV) mutants depends on their potential resistance to antiviral therapy, enhanced virulence, and alteration of viral epitopes important for the host immune response. The importance of the HBV polymerase gene (p-gene) mutants may depend on alterations of the hepatitis B surface antigen (HBsAg) protein carrying neutralization epitopes. The biology of HBV infection caused by monoclonal population of HBV p-gene and viral envelope (s-gene) mutants is not well characterized. Pathogenetic and molecular studies of the infection induced by monoclonal HBV mutants engineered in vitro in chimpanzees may contribute to a better understanding of the significance of HBV mutants for hepatitis B diagnosis, treatment, and prevention.

The HBV p-gene mutant virus with three amino acid mutations (rtV173L, rtL180M, rtM204V) and monoclonal s-gene mutant sG145R were prepared by a PCR method using site-directed mutagenesis. The HBV mutants were intravenously inoculated, each in two chimpanzees, and serial serum samples from the animals were tested for HBV DNA, HBsAg, antibodies to hepatitis B core antigen (anti-HBc) and surface antigen (anti-HBs), and alanine aminotransferase (ALT) activity. Liver biopsies, collected weekly, were tested for histopathology and for the presence of HBsAg and hepatitis B core antigen (HBcAg). Molecular characterization of the mutation sites in p-gene and in s-gene was carried out by sequencing of multiple clones of polymerase gene or a portion of s-gene containing the a-determinant from serum samples positive for HBV DNA.

Intravenous administration of the HBV p-gene mutant preparation resulted in viremia marked by the presence of HBV DNA in serum between week 1 and 5 post-inoculation in both chimpanzees. HBsAg was detected in only a single serum specimen (day 31 post-inoculation) by using a commercial assay (Auszyme® Monoclonal, Abbott). Sequencing of multiple HBV DNA clones from the first and last HBV DNA-positive serum samples showed that all three mutations in p-gene were present throughout the period of viremia in both chimpanzees. Seroconversion to anti-HBc and anti-HBs was observed during week 6 or 8 postinoculation, respectively, in both animals. Neither of the two chimpanzees inoculated with HBV p-gene triple mutant had an elevation of the ALT activity level above the normal cut-off value during 185 days of the follow up period. In one of the two sG145R-inoculated chimpanzees, HBV DNA appeared in serum 14 weeks after inoculation and lasted until week 22. HBsAg was detected in serum from week 17 to 24. Sequencing of the first HBV DNA positive serum from this animal showed a mixed population of mutant and wild-type viruses with a majority (67% of the clones) of the viral population reverted to wild-type sequence. Analysis of a subsequent serum sample showed a complete reversion to the wild-type sequence; no G145R mutant was detected in any of the 29 clones sequenced. Anti-HBc was detected from week 21 and anti-HBs from week 25. ALT was elevated with a peak characteristic for acute hepatitis (5x cut-off value) from week 21 to 23. The second sG145R-inoculated animal was positive for anti-HBs only, from week 9 to 12 post-inoculation.

Experimental infectivity data showed that chimpanzees inoculated with monoclonal genetically engineered polymerase triple mutants developed HBV infection with preservation of the mutations in the polymerase gene throughout the duration of the infection. Inoculation with monoclonal G145R mutants induced HBV infection with a biochemical pattern of acute hepatitis that was accompanied by a reversion to wild-type sequence. The virologic characteristics of the HBV infection without liver pathology induced in chimpanzees by HBV p-gene triple mutants and acute hepatitis observed in the chimpanzee infected with the s-gene mutant reverting to wild-type virus may be attributed to the dynamics of molecular mutations in the replicating virus, the size of the administered inoculum, or the replication fitness of the HBV mutants.

Interferon alfa was licenced by the FDA in 1992 as the first treatment for chronic hepatitis B following successful trials with interferons and unacceptable toxicity in trials with the antiviral agent adenine arabinoside. Subsequently the development of the antiviral lamivudine suggested that the side effects of interferons may disadvantage them despite their superior efficacy. However viral resistance due to mutations has hindered lamivudine and a recent EASL consensus meeting recommended interferon as first line therapy for moderate or severe chronic hepatitius B. Newer antivirals also have potential for viral resistance although perhaps less frequently. Since the pathogenesis of HBV is immune mediated, efforts to lead to HBeAg seroconversion have involved immune therapies. Other immune agents such as IL12 were not effective. Therapeutic vaccines have considerable promise but are still in trials. The Cytotoxic T cell (CTL) response to HBV peptides is vigorous and multispecific following acute hepatitis in contrast to chronic hepatitis B where it is weak or non-existent. However the use of Dendritic cells, the professional antigen presenting cells, produced in vitro by the use of IL 4 and GM CSF and HBV peptides, has been able to reverse this anergy in vitro. Despite their promise this would be very labour intensive in vivo. The development of the pegylated interferons and their superior efficacy in chronic hepatitis C has lead to their application in chronic hepatitis B. There have been several trials to date. The first study used the larger Peginterferon (Peginterferon alfa2a 40Kd) for 6 months in 193 patients in the Asian Pacific region and showed superior efficacy when compared with conventional interferon alfa2a. HBeAg seroconversion rates were 37% and 33% at 90mg and 180mg Peginterferon compared with 25% with conventional interferon. Peginterferon gave better results in difficult groups -High DNA, ALT<2xULN, and Genotype C. A preliminary report of a trial from Hong Kong using Peginterferon alfa2b (12KD) in 40 patients with HBeAg positive disease suggested combination of Peginterferon and lamivudine was superior to Peginterferon alone. However a larger European study of 266 patients (20% Asian) receiving Peginterferon Alfa2b (12KD) in combination with lamivudine for 12 months or Peginterferon alone showed no difference 6 months after stopping therapy for HBeAg seroconversion (36% vs 35%), DNA<200,000 (27% vs 32%), or normalization of ALT (32% vs 35%). Similarly, a multinational study of 537 HBeAg negative patients showed no benefit of combination therapy of Peginterferon alfa2a(40KD) and lamivudine for 12 months compared with Peginterferon alone 6 months after stopping therapy with 60% and 59% of patients having normal ALT and DNA<20,000 copies in 44% and 43%. Results with lamivudine were lower at 44% for normal ALT and 29% DNA <20,000. In conclusion, Peginterferons have superior efficacy than conventional interferon or lamivudine. Combination of lamivudine and Peginterferon may be no better than Peginterferon alone. The pendulum of antiviral agents or immune agents appears to be swinging back towards interferons (PEG) especially in younger patients.

Of the two antiviral nucleoside/nucleotide analogues that are licensed, lamivudine is limited by the incremental development of resistant hepatitis B YMDD mutants 1 and adefovir by the potential renal toxicity, 2 so that the current dose being licensed for adefovir is the minimal effect dose for the hepatitis B virus. Entecavir, a guanosine analogue, has a strong inhibitor effect of the priming of HBV polymerase by guanosine triphosphate. 3 No entecavir-resistant mutants were detected in woodchucks after 3 years of treatment. A phase 2 trial 4 shows that at 6 months, both 0.1 mg and 0.5 mg daily doses of entecavir are more effective than 100 mg lamivudine in viral suppression. Entecavir, at a higher dose of 1 mg daily, is also effective against YMDD mutants. 5 Telbuvidine is one of three L-nucleosides with specific HBV inhibitory activity. 6 Like lamivudine, which is also an L-nucleoside analogues telbuvidine has almost no side-effects. A phase 2 trial 7 shows that at 12 months, telbuvidine, either alone or in combination with lamivudine, causes significantly better viral suppression than lamivudine monotherapy. Telbuvidine 400 mg daily and 600 mg daily both cause an unprecedented reduction in median HBV DNA concentrations of more than 6 1og 10 at 12 months' treatment.

Tenofovir disoproxil fumarate, structurally similar to adefovir and approved for treatment of HIV, is also effective in suppressing replication of YMDD mutants. 8,9 However, no studies have been done in patients with the HBV infection alone.

Another compound LB80380, a phosphonate nucleotide analogue, of guanosine monophosphate, is also active against the YMDD mutants in vitro. A phase I/IIa trial in humans shows ~3.8 log suppression of HBV DNA after 4 weeks of therapy.

The option of combining two (or more) nucleoside analogues, as in HIV therapy, is theoretically sound. It keeps viral suppression to a maximum, thereby reducing the chance that resistance will develop. 6 Moreover, the nucleoside analogues that are licensed or under trial have few side-effects. Preliminary results of the combination of telbuvidine and lamivudine in a phase 2 trial show no additional benefit over telbuvidine monotherapy. 7 A similar trial of the combination of adefovir and lamivudine also showed no additional benefit over lamivudine monotherapy. 10 Phase 3 trials of other combinations of nucleoside analogues have yet to be started and might show exciting results.

Maximal, long-lasting suppression of HBV replication is the key to achieve the clinical goals of treatment in patients with chronic hepatitis B. The theoretical advantages of combination therapy are greater suppression of viral replication, increased efficacy in restoring HBV-specific T-cell reactivity and a lower rate of drug resistance. By targeting more than one site in the replicative cycle of HBV and/or the host immune response, the combination regimens are expected to increase the rate of complete treatment response (HBsAg clearance/anti-HBs seroconversion). Two principle approaches have been investigated: i) antiviral plus immunomodulator, or ii)a combination of nucleoside/nucleotide analogues. Contrary to the expectations, the combination of nucleoside/ nucleotide analogues did not achieve higher suppression of HBV in comparison with monotherapy, however, it may reduce the rate of drug resistance.

Currently, there is insufficient evidence to decide which patients will benefit from combination therapy; what agents to include, how to plan the timing of the components in a combination regimen. One major issue is to have a better definition of different patients' subgroups. The application of new technologies such as analysis of HBV kinetics during treatment, monitoring HBV-specific T-cell reactivity, proteomics and differential gene expression by DNA microarrays will provide the evidence base for developing different combination treatment regimens. The second major development will be new antivirals and more specific immunomodulatory approaches.

Chronic hepatitis B is a complex and heterogenous disease entity and the challenge for the future is to design individually tailored combination treatment regimes, which will be safe and much more effective in achieving complete treatment response.

Pathogenesis of hepatitis B virus (HBV) infection is typically the result of the outcome between the three-way interaction of virus-hepatocyte-host's immune response. Over the natural history of chronic hepatitis B (the immune tolerance phase; the immune elimination phase; the non-replicative phase), the disease spectrum covers chronic hepatitis, cirrhosis and hepatocellular carcinoma. In the context of therapeutic intervention, for each antiviral drug selection pressure (eg. lamivudine, adefovir, entecavir) that is introduced, escape mutants are selected from the pre-existing pool of quasispecies. In the clinical context, the emergence of drug-resistant HBV has now been associated with worsening liver function, progressive liver disease, serious acute exacerbations of hepatic flares, hepatic decompensation and even death. In immune compromised patients, this process is often rapidly accelerated, presumably due to the higher replication rates as well as lack of immunological control. Antiviral drug resistant mutants emerge as a function of at least six factors: the viral mutation frequency, the intrinsic mutability of the antiviral target site, the selective pressure exerted by the drug, the magnitude and rate of virus replication, the overall replication fitness of the mutant and the availability of replication space. All of the drug-specific mutations for each agent have been mapped to the viral polymerase (Pol). Because of the overlap of the reading frames of the HBV Pol with the frame-shifted hepatitis B surface antigen (HBsAg), drug-resistant mutations in the HBV Pol can directly impact on the nature of HBsAg and its function, including properties of viral neutralization. Drug resistant HBV can behave as a vaccine escape mutant and cases of transmission of drugresistant HBV have now been reported. Clearly, improved treatment strategies are urgently required to prevent the continued selection of HBV drug-resistant mutants, otherwise inadequately treated patients and the wider community will suffer the subsequent disastrous consequences. Table 1 Nucleoside Analogues

Orally available 

Hepatitis due to hepatitis B virus (HBV) reactivation is a serious cause of liver-related morbidity and mortality in hepatitis B surface antigen (HBsAg) positive patients undergoing cytotoxic or immunosuppressive therapy. As the hepatitis is related to HBV virological reactivation, application of effective anti-viral therapy to HBV (anti-HBV) such as lamivudine and famciclovir has been attempted. Despite the use of these anti-viral agents at the time of clinical hepatitis, some HBsAg positive patients still developed hepatic failure and died. This is probably related to the late institution of the nucleoside analogues when the immunemediated liver damage has already been established. Hence, it is now generally accepted that nucleoside analogues should be administered pre-emptively before the onset of hepatitis due to HBV virological reactivation. Our recent data suggested that "early" is preferable to "delay" pre-emptive therapy to initiate lamivudine in these settings. Currently, there are a number of new nucleoside analogues such as adefovir, entecavir, emtricitabine, clevudine, b-L-thymidine, b-L-Fd4C or non-nucleoside analogues such as the phenylpropenamide derivatives AT-61 and AT-130, in the anti-HBV pipeline. Further clinical trials with the use of these nucleoside analogues will be needed to determine their antiviral efficacy in the hepatitis B patients receiving immunosuppressive therapy. On the other hand, identification of risk factors for hepatitis B patients undergoing immunosuppressive therapy would allow one to be more selective in the use of pre-emptive nucleoside analogue therapy.

Hepatitis B virus (HBV) infection has an adverse effect on the clinical outcome after kidney transplantation. Liver complications in immunosuppressed HBsAg-positive renal transplant recipients can manifest as potentially fatal acute hepatitic exacerbations, chronic liver disease, or hepatocellular carcinoma. Difficulties in predicting the clinical course of liver disease in individual patients confound clinical management. Nucleoside analogue therapy results in effective suppression of HBV replication and improved clinical parameters of liver disease, but the effect on patient survival and the risk of drug resistance remained obscure. Quantitative HBV DNA assays allow earlier detection of increased viral replication, before the onset of biochemical abnormality. We have combined serial HBV DNA monitoring with pre-emptive lamivudine therapy, so that kidney transplant recipients with increasing levels of HBV DNA will be started on lamivudine therapy. Our results showed that this strategy markedly improved patient survival to simulate that of HBsAg-negative subjects. Improved liver biochemistry was noted in all patients who had elevated transaminase levels at the time of starting treatment. Prolonged treatment was however associated with the selection of drug resistant YMDD variants. Hepatitic flares were common after the development of drug resistance, and could lead to decompensation in a small proportion of patients. With careful selection, based on negative HBeAg status, normal liver biochemistry, and the absence of recent increase in immunosuppressive dose, discontinuation of anti-viral treatment was feasible in about one-fifth of treated subjects.

Combining serial HBV DNA quantitation with early lamivudine therapy is an effective way to reduce liver complications and improve the survival of HBsAg-positive renal allograft recipients.

The lamprey is amongst the most ancient of vertebrates. During its growth and development, there is a sudden, dramatic and complete arrest in the the biliary epithelial cell lineage such that the adult liver is without a bile drainage system.. This 'biliary atresia' leads to an interesting pathway in the lamprey of excreting bile, including bilirubin, which when oxidized, becomes biliverdin.

This dissociation of heptocytic and biliary cell lineage specificity, differentiation pathways and cell fate raises intriguing questions on the ontogeny of the cellular elements in the liver. The heterogeneity and plasticity of stem cells in the liver is discussed.

Hepatocytes and biliary epithelial cells originate from a common endodermal foregut bud. These cell lineages can be sustained by the growth of mature (unipolar, terminally differentiated) hepatocytes or biliary cells; or from a bipolar ductular progenitor cell (oval cell). There is another multipotential periductular progenitor cell, presumed to be from bone marrow origin. The dynamic interchange between and amongst these subpopulations of cells with proliferative capacity is not clearly understood but its understanding holds the key to the secret of liver regeneration, or failure to do so. It will also shed light on the carcinogensis of hepatocellular cancer and cholangiocarcinomas.

Finally, a provocative hypothesis, which stipulates that the transdifferentiation of mature cells (hepatocytes and biliary ductal cells) is possible. This challenges the accepted paradigm (or dogma) of cell growth and differentiation, and may open a new field to cell and tissue engineering. It may also explain why lampreys are green.

In this lecture I will review the principles of viral dynamics and show how they can be used to evaluate the effectiveness of drug therapy. I will also point out the need to consider pharmacokinetics when therapy involves pegylated interferon a2b. Lastly, I will show how viral kinetics can provide insights into the events occurring during re-infection of the liver after a liver transplant.

Liver Diseases Section, NIDDK, National Institutes of Health, Bethesda, MD, USA Clearance of the hepatitis B virus (HBV) requires a coordinated innate and adaptive, humoral and cellular immune response. In acute, self-limited hepatitis, most HBV-DNA molecules are cleared in the incubation phase, i.e. prior to the onset of liver damage and clinical symptoms of acute hepatitis B. This rapid reduction in viral load is attributed to inhibition of viral gene expression and replication by cytokines such as IFN-g and TNF-a, which are detectable in the liver even prior to infiltration of large numbers of HBV specific T cells. A vigorous, polyclonal and multispecific Th and CTL response then coincides with maximum ALT elevation, clearance of HBe and HBs antigens and development of neutralizing antibodies. After recovery from hepatitis B, HBV specific T cells as well as neutralizing antibodies persist for decades. Despite of serological clearance of all viral antigens, however, trace amounts of HBV-DNA remain detectable in serum and PBMC suggesting a balance between CTL-and antibody inaccessible viral reservoirs and de novo induced immune cells that control viral spread.

Persistently infected, HBeAg-, HBsAg+ patients have also recently been shown to mount functio-nal HBV specific CD8+ T cell responses in blood and liver that allow viral control without liver in-jury. In contrast, in HBeAg+ HBsAg+ patients with high viral load and an extensive intrahepatic in-flammatory infiltrate, the number of circulating HBV specific CD8+ T cells is generally below the de-tection limit of sensitive tetramer techniques and in the liver, T cells of a given HBV epitope specifici-ty are more diluted among other inflammatory cells. Importantly, several studies indicate that T cell responsiveness can be spontaneously or therapeutically restored in chronically infected patients con-comitant with a reduction of viral load and/or HBsAg clearance.

In summary, these results demonstrate that acute and chronic hepatitis B should be regarded as extremes of a biological continuum rather than distinct entities and may open avenues for immunomodulatory therapies.

A number of factors are important in the pathogenesis of alcoholic liver injury. These factors include endotoxin, cytokines and other pro-inflammatory mediators, oxidative stress and immunological mechanisms. Multiple lines of evidence support the hyprothesis that endotoxin or lipopolysaccharide (LPS) is an important cofactor in the pathogenesis of alcoholic liver disease (ALD).

There is a strong correlation between the level of endotoxemia and the degree of liver injury. Kupffer cells are important in clearing LPS but it's interaction is not limited to clearing LPS. Kupffer cells when exposed to LPS can be activated to produce a spectrum of cytokines and reactive oxygen intermediates.

Multiple mammalian receptors for endotoxiin have been identified and they include two major glycoproteins: LPS -binding protein (LBP) and CD14. Levels of CD14 in Kupffer cells correlate with the severity of liver injury in experimental animals and humans. Recently members of the toll-like receptor family have been implicated in LPS signaling and TLR4 deficient mice are protected from liver injury.

One major mediator of the effects of oxidative stress and endotoxin is nuclear factor kappa B (NF-kb). Among the genes activated by NF-KB are several proinflammatory cytokines such as tumor necrosis factor alpha, chemakines and cyclooxygenase-2.

In ethanol-fed rats, increased expression of cox-2 occurs in association with endotoxemia and oxidative stress. Increased oxidant stress is due to an imbalance between increased levels of pro-oxidants (iron and cytochrome P4502E1) and decreased levels of antioxidants such as the antioxidant enzymes and vitamin E. Other factors that are important in ALD are increased apoptosis, enhanced fibrogenesis which eventually leads to cirrhosis.

Hepatic encephalopathy (HE) is a major neuropsychiatric complication of liver failure and the precise mechanisms involved in its pathogenesis are not fully understood. Neuropathologic studies of brain tissue from patients who died in hepatic coma show swelling of mitochondria and margination of chromatin in non-neuronal elements, particularly glial cells (astrocytes). Novel insights into pathophysiologic mechanisms continue to be provided by molecular techniques such as differential display / RT-PCR and gene chip microarrays. Using such techniques, alterations in expression of genes coding for essential brain proteins have been reported in both experimental and human HE. Such proteins include the astrocytic structural protein GFAP, the glial glutamate and glycine transporters EAAT-2 and GLYT-1 and the mitochondrial "peripheral-type" benzodiazepine receptor (PTBR). Exposure of primary glial cell cultures to liver-derived toxins such as ammonia and manganese results in a similar pattern of alterations of gene expression. Gene "knock-down" of EAAT-2 leads to brain edema and hyperexcitability, features which are characteristic of liver failure. Loss of expression of EAAT-2 and GLYT-1 in experimental liver failure leads to increased extracellular brain concentrations of the neuroactive amino acids glutamate and glycine; increased PTBR mRNA, on the other hand, results in increased synthesis of neurosteroids such as allopregnanolone with potent neuroinhibitory properties. Accumulation in brain of these neuroactive substances could contribute to the pathogenesis of HE in acute and chronic liver failure [funded by the CIHR Canada].

HCC (hepatocellular carcinoma) is one of the most important medical problems facing the Asian Pacific region where we have so much chronic hepatitis B and C virus infection. As an indication of the magnitude of the problem, we have approximately 500 admissions due to this malignancy every year in our Department of Gastroenterology at the University of Tokyo. It is of utmost importance that we have efficient screening, diagnosis and treatment available. Over the last several years, there has been steady improvement in elucidating the group at super high risk of developing hepatocellular carcinoma, and in treatment modalities for the management of these patients.

At this meeting, I will discuss how to elucidate the super-high risk group of developing HCC by studying molecular as well as clinical features of patients suffering chronic B-and C-viral liver diseases. We set-up the way of analysing RNA profiling of neoplastic and non-neoplastic tissue by cloning and sequencing full length cDNAs obtained from various liver tissues. We also analysed SNPs (Single Nucleotide Polymorphism) to elucidate underlying genetic backgrounds and found several responsible foci.

In addition, there is a simple measure to set up a high risk group. I found platelet count correlate with the degree of hepatic fibrosis, namely, 70,000/mul often found in patients with F1, 150,000 in F2, 130,000 in F3, and below 100,000 in F4 (cirrhosis). By simply counting the platelet count, we can identify patients in whom extensive screening should be performed. By defining high risk by platelet count, screening by AFP, AFP-L3 and DCP, and by imaging techniques, we may improve the patients' 5-year survival rate to 70%.

The pathogenesis of human liver disease is characterised by the broad pathological processes of inflammation, fibrosis, cellular proliferation and apotosis. We have used gene array analysis to study the following diseases 1.

Hepatitis C associated cirrhosis (with and without HCC) 2.

Hepatitis B associated cirrhosis under antiviral therapy 3.

Autoimmune diseases (Autoimmune hepatitis, Primary Biliary Cirrhosis and Primary Sclerosing Cholangitis) 4.

Early chronic hepatitis C infection 5.

HCV infection post liver transplantation 6.

Alcoholic Liver Disease (human and chimpanzee)

The data generated has enabled us to highlight significant pathways that had been under-recognised in human liver injury. These include 1.

The dominance of the TH1 profile in hepatic inflammation associated with chronic hepatitis C infection 2.

The identification of Drosophillia related genes particularly the wnt pathway in all forms of human cirrhosis 3.

Identification of the unique molecule RER in chronic hepatitis C infection 4.

The identification of many protoncogenes in hepatitis C cirrhosis associated with hepatocellular cancer 5.

Identification of a molecules such as Emprin, DDR1 and Claudin 10 as important molecules associated with hepatic fibrosis in chronic hepatitis C infection 6.

The identification of a strong antiviral response. (Interferon alpha associated transcripts) in hepatitis C infection.

The replication of hepatitis B virus (HBV) is a well-organized process, which involves alternating use between DNA and RNA in the viral genome. The viral DNA polymerase which plays a major role in this process is composed of four domains and starting from the N-terminus they are the terminal protein (TP), a spacer region which has not been associated with a specific function, the reverse trasncriptase (RT) and the RNase H, which digests the RNA in RNA-DNA hybrids. Since HBV has its own replicating polymerase, a number of nucleoside analogues have been developed as antiviral drugs targeting at this enzyme. The precise targets of HBV polymerase are at the YMDD motif of the C domain, which is at the catalytic site, or at the B domain in the RNA-dependent RNA polymerase. Two major approaches have been used to analyze the functions of HBV polymerase. One is the molecular approach, which is to establish cloning and expression systems for direct analysis of the polymerase function without involving other HBV viral proteins. The other is to study the functions of polymerase in naturally occurring full-length viral genomes isolated from patients. Employing the second approach we have reported two full-length HBV genomes with high homology in nucleic acid sequences (98.7%) but replicated at high and low virus loads (#56, high replicative, #2-18, low replicative) in patients. To study the structural basis for this difference in replicative competency, chimeric constructs were generated by substitution of the TP, spacer, RT and RNase H genes in #2-18 by those from #56 respectively. Replicative competency was studied using these chimeric constructs by cell transfection, and only when residue 652 was changed from serine to proline, the replicative competency could be enhanced. Compared to the RT model of HIV, computer modeling of HBV RT suggested that residue 652 is located at the connecting loop or the hinge between the two helices of the thumb domain of RT. Since these two helices were predicted to form part of a translocation tract, which controls the relative movement of the polymerase, the rigidity of residue 652 constrains the formation and/or position of the two helices. Proline is more rigid, while serine is more flexible. A change from proline to serine at residue 652 could disrupt the relative conformation of the helices and thus would affect the polymerase activity (J Virol, 2001 75:11827).

To further identify the importance of residue 652 in HBV replication, proline at residue 652 in #56 was mutated to 11 other amino acids (phenylalanine, arginine, glycine, alanine, threonine, tyrosine, asparagines, glutamine, valine, leucine and serine). These mutants were separately used to transfect HepG2 cells, and replicative competencies of these mutants were assayed by real-time PCR. Results showed that changes in the hydrophilicity of amino acid at residue 652 did not affect the replicative competency. While, when proline was mutated to glycine, which has a simple structure, which could confer the hinge with higher flexibility, significant decreased replicative competency was observed. In contrast, when proline was mutated to amino acids with hydroxyl groups (Asp, Glu), the replicative competency of these mutants was higher than that of the wild-type virus. The highest replicative competency was found when proline was changed to glutamine, which suggests that the number of hydroxyl groups and the length of side chains in the structure of amino acid at 652 are associated with high replicative competency. The mechanisms of the enhanced replicative competency could possibly be due to formation of hydrogen bonds of the side chains of glutamine with other adjacent amino acids to constrain the conformation of the helices, resulting in higher replicative competency.

In conclusion, the replicative competencies of these site-directed mutants further support that amino acid 652 in RT is an important residue regulating the replicative competency of HBV strains, and the hinge region of RT in HBV could be a target for development of novel anti-HBV drugs.

KEYNOTE LECTURE 10 -TRANSPLANTATION AND LIVER DECOMPENSATION

Baylor University Medical Center and Baylor Regional Transplant Institute, Dallas, Texas, USA Chronic hepatitis C is the leading indication for liver transplantation (LT). The proportion has grown steadily since 1991 and now accounts for over 40% of LT. This trend is projected to continue for another 2 decades. HCV infection recurs in all patients after LT and results in fibrosis in half after 5 years. The severity of recurrent HCV after LT may be related to the level of HCV RNA at the time of LT. Antiviral treatment before LT may decrease HCV RNA and reduce the risk of or severity of recurrence, but is difficult to administer. Many patients have contraindications to therapy and about 80% of those treated require dose reductions. Growth factors may help prevent dose reductions. Nonetheless, eradication of virus during therapy occurs in 20-40% and recurrence after transplantation may not occur in these cases. The severity of HCV recurrence appears to be worse if the donor is elderly or living, and consideration must be given to excluding such donors. Immunosuppression after LT accelerates liver injury. Thus, preemptive strategies to reduce the incidence or severity of recurrence have been developed including immunoglobulin, early antiviral treatment, and rapid steroid withdrawal. Pilot studies with neutralizing antibodies directed against HCV have not been encouraging, but the doses studied so far are low. Early interferon treatment when HCV levels are low and clinical disease is not yet apparent might prevent later graft injury. A large study of pegylated interferon and ribavirin is about to start, but drug tolerance during the early weeks after LT will be problematic. Treatment of established recurrence of chronic hepatitis after LT is difficult since most patients have genotype 1, high HCV RNA levels, and some degree of fibrosis. Furthermore, anemia and cytopenia are common because of the effects of medications and pre-existing hypersplenism. Low dose treatment must be started cautiously and increased as tolerated. Dose reductions are required in about 70%, but sustained viral clearance is possible in 20-30%. In summary, interferon-based regimens are the only available option for treating HCV in the transplant setting but therapy is difficult and response is much less than in non-immunosuppressed patients. Certainly, better strategies are needed to control HCV infection in the transplantation setting.

The current paradigm of transplant immunology envisions that the recipient's immune system is left more or less intact and the transplanted organ is viewed as a source of continued antigenic stimuli, which would require lifelong immunosuppression to prevent rejection. However, the finding that following treatment of vigorous allograft rejection, subsequent reversal of the rejection process and a diminution of the need for high dose immunosuppression suggests that a dynamic balance between the allograft and recipient immune response occurs, and in some patients can lead to a drug-free state without allograft injury. The stability of allograft viability is most pronounced in liver transplantation. This was demonstrated in a prospective protocol of immunosuppressive weaning in long-term liver transplant survivors at our center.

Recently, it has been suggested that alloreactivity is a necessary condition for the development of tolerance and it has been suggested that early heavy immunosuppression, while preventing early graft loss, may also eliminate the necessary step of immune activation that is required for clonal exhaustion/deletion. While our approach to long-term immunosuppression is a minimalist one, we have incorporated the principle of individually measured response to post-transplant immune activation to the allograft into our immunosuppressive regimen, with the aim to achieve the right balance of preconditioning and minimal short-term monotherapy immunosuppression. It is our aim to initiate earlier weaning and may result in a stable drug-free, or tolerant, organ recipient.

The two therapeutic principles of this approach include recipient pretreatment and the use of minimal post-transplant immunosuppression. Preconditioning is done with a peri-operative recipient lymphoid depletion with anti-lymphocyte antibodies, followed by monotherapy with tacrolimus. Evidence of immune activation on allograft biopsies is not treated unless there are histopathological findings of immune destruction and clinical correlation. After a period of clinical stability, dose reduction of maintenance immunosuppression is started -this has led to dramatically lowered dosing of tacrolimus with its associated benefits, such as improved renal function and absence of diabetes.

It has been customary for physicians to use high dose and multiple immunosuppressive agents to prevent rejection of the transplanted organ, beginning at the time the organ is implanted. However, there is abundant experimental data, which suggests that elimination of the all-important, initial immune reaction against the donor organ, will inhibit the process of clonal exhaustion/ deletion. Without this activation, T cells targeted against the graft will persist, making it forever necessary that patients rely on drugs to protect the organ from being rejected. Thus, we are proposing, even though it may seem counterintuitive, to allow the immune activation against the donor organ to occur, but in a subdued fashion so that the clonal deletion of the T cells is both more complete and long lasting.

The results of liver transplantation (LTx) in patients with chronic hepatitis B virus (HBV) infection has significantly improved as a result of the rapid evolution in the strategies of postoperative prophylaxis. Hepatitis B immunoglobulin (HBIg) is the first effective prophylactic agent but the drug is costly and may not be completely effective. The initial success of lamivudine monoprophylaxis was overshadowed by concerns with the emergence of viral mutants and combination therapy adding HBIg to lamivudine has become a widely adopted approach. The newer nucleoside analogs such as adefovir appear most promising and a combination of two or more antiviral agents may be the future prophylactic strategy of choice. The current HBV prophylactic regimen at Queen Mary Hospital focuses on nucleoside analogs and consists of lamivudine monotherapy with adefovir reserved for breakthrough reinfection before or after LTx. With the extended experience of 176 patients who underwent LTx with lamivudine monoprophylaxis, the overall patient survival was 89.2% at a median follow-up of 23 months. Fifteen (8.5%) patients developed breakthrough reinfection with YMDD mutant which was effectively controlled with adefovir. Since lamivudineresistant mutants were more common in HBV genotype C than genotype B (2-year mutant rate 23.6% vs 0%, p=0.009), it may be worthwhile to consider adding adefovir to lamivudine prophylaxis in patients with genotype C. Our recent report on observation of active production of anti-HBs after LTx suggests the possibility of adoptive transfer of immunity against HBV from an immune donor through a liver graft. A novel strategy of active immunisation of a living donor before transplantation is currently under investigation. The idea of active immunisation after transplantation is also attractive but the results have been conflicting to date. The most cost-effective prophylactic strategy remains to be defined. 

The clinical and follow-up data of 24 patients with HCC with PVTT receiving OLT (transplant group) from Feb. 1999 to March. 2003 were analyzed and compared to that of 27 patients undergone routine hepatic resection (resection group) and 59 patients without surgical treatment (non-surgical group).

The perioperative mortality ( one month ) was 0% for transplant group. 

OLT is an effective but palliative treatment modality for HCC patients with PVTT followed with prolonged survival but poor tumor free survival rate. The lifetime prevalence of varices in cirrhotics has been reported to be as high as 80-90%. Esophageal varices are present in at least 50% of compensated cirrhotic patients at diagnosis. Severe upper gastrointestinal (UGI) bleeding as a complication of portal hypertension develops in 30-45% of cirrhotics.

Major predictors of variceal hemorrhage are high risk varices (> 5 mm in size and presence of red color signs), severity of liver disease and high (>16 mm Hg) hepatic venous pressure gradient (HVPG) or intravariceal pressure (measured non-invasively by a pressure gauze). Patients with a high HVPG have a high risk of poor evolution, and may be candidates for more intensive and aggressive therapy, such as surgery or TIPS. The pharmacological treatments have been aimed at reducing the HVPG and correcting the increased portal blood inflow by the use of splanchnic vasoconstrictors, such as beta-blockers, vasopressin derivatives and somatostatin.

The initial episode of acute variceal bleeding should be managed with endoscopic therapy in combination with vasoactive agents, such as terlipressin (2 mg every 4-6 hr.), somatostatin (250 to 500 mcg/hr infusion) or octreotide (50 mcg/hr infusion). However, high rebleeding rates in patients with advanced liver disease is common. It is not clear whether giving vasoactive agents for 2 or 3 days is as effective in preventing rebleeding as the current practice of giving 5 day therapy. Control of infection with early adequate antibiotics and restrained replacement of blood transfusion additionally help in the prevention of variceal bleeding. With the current combination therapy protocols, success can be achieved in 90-95% of patients with acute variceal bleeding. Patients who bleed again after the first-line therapy, and those with persistent risk factors should be considered for variceal decompression with transjugular intrahepatic portosystemic shunt (TIPS) or surgical shunt. Devascularization procedures are reserved for patients who are not candidates for decompression because of venous thrombosis. For patients with end-stage liver disease, liver transplantation may be the most appropriate treatment option.

Prevention of variceal rebleeding is another major objective in the management of variceal bleeding. Endoscopic therapy has been found to be quite successful in variceal obliteration and prevention of variceal bleeding. Endoscopic variceal ligation (EVL) a technically easier procedure than EST has been shown to be more effective in the prevention of rebleeding. Controlled trials have shown that EVL is superior to EST as it achieves variceal obliteration quicker, in fewer treatment sessions and also has inherently lower complication rates. However, endoscopic therapy does not reduce the portal pressure. There is sufficient evidence to support the use of combination of beta-blockers and nitrates as an equally effective and safe alternative to endoscopic therapy of variceal bleeding.

The mortality rate from first UGI bleed remains very high (20-35%) despite aggressive management. This makes primary prophylaxis, or prevention of initial variceal bleeding an important therapeutic goal. The options currently available for the same are beta-blockade, and endoscopic variceal ligation (EVL). Non-selective beta blockade has been reported to be effective in prevention of first variceal bleed. There are however multiple problems in the use of beta-blockers. Around 15% of the patients have contraindications, another 15-20% experience side effects to beta-blockers. Of the remaining, at least 25-30% patients do not achieve adequate reduction in portal pressure to the level required to prevent bleeding. Other problems with beta-blockers are the lack of patient compliance, need for lifelong therapy and the increased risk of bleeding after the cessation of therapy. EVL has been shown to be better than beta-blockers in primary prophylaxis in a previous trial from our center. In recent meta analyses, EVL reduced the risk for first variceal bleed but had no effect on mortality as compared to beta-blockers. It has been observed in very recent studies that while a combination of EVL and beta-blockers is as effective as either of these treatments alone; the combination is superior in prevention of variceal recurrence.

The key to improved survival remains in the prevention of enlargement of varices, the early primary prophylaxis. The current data from multicentric studies form the US however, did not support the use of BB therapy in these patients. Pre-primary prophylaxis (prevention of formation of varices) is a far fetched goal and whether all cirrhotics could be put on drug therapy to prevent development of varices and other complications remains an unresolved issue.

Gastric varices (GV) are observed in approximately 20% of all unselected patients with portal hypertension. They are more common in patients who have bled than those who have not bled. About 25% of GV bleed during lifetime. Gastric variceal bleeds are often more severe and associated with high mortality.. Frequency and severity of bleeding from GV depends on their location which forms the basis for their classification. Gastric varices could be associated with esophageal varices {Gastroesophageal varices (GOV): type 1 (GOV1) -along the lesser curve or type 2 (GOV2)-along the fundus, or are present in isolation {Isolated gastric varices (IGV); in fundus -(IGVI) or at ectopic sites in the stomach or the first part of duodenum (IGV2)} . Gastric varices could be primary (at first presentation) or secondary (appearing after obliteration of oesophageal varices). IGV1 and GOV2 type of GV bleed more often (78% and 54% respectively) and more massively than the lesser curve varices.

Several modalities have been tried to control bleeding from gastric varices with variable success. In nearly 60% of the patients with GOV1, the varices disappear when the oesophageal varices are obliterated. Hence they do not require specific therapy. Injection of cyanoacrylate glue is highly effective in patient with GOV2 and IGV1. On the other hand, patients with ectopic varices, IGV2 rarely bleed and can also be managed with glue injection if endoscopically approachable. Balloon-occluded retrograde transvenous obliteration (BRTO) is a radiological modality for controlling bleeding from gastric varices and for recurrent encephalopathy due to spontaneous shunts.

Portal hypertensive gastropathy (PHG) is another important cause of upper gastrointestinal bleeding in patients with portal hypertension. Prospective studies have reported that PHG is present in more than 50% of cirrhotics. PHG developing after variceal eradication is often transitory and clinically insignificant. However, if PHG is pre-existing, endoscopic therapy for varices could worsen the PHG, with a likelihood of bleeding. In such patients, concomitant beta-blocker therapy should be initiated.

In summary, the management of variceal bleeding has improved significantly in the last decades. Availability of a large armamentarium has allowed us to use and evidence based rational approach leading to better prevention of variceal bleeding, higher bleed control with improved survival. Newer therapeutic targets based on improved understanding of the pathogenesis of portal hypertension are also promising.

Since the prognosis of hepatocellular carcinoma (HCC) is extremely poor in patients with advanced HCC, the early detection of HCC is important for an effective treatment. Screening tests for HCC in the high-risk population can detect tumors at an earlier stage and thus confer a higher chance of receiving treatment.

To date, the screening of HCC has been performed in the high-risk population. However, no randomized controlled studies demonstrating improved survival still remain some arguments regarding the efficacy of screening. The Practice Guidelines for the effective screening of HCC should be established according to the individuals possessing different risk factors.

For an effective national screening program for early detection of HCC, we analyzed the risk factors for HCC through a 9 year prospective study from 1990 to 1998, of 4339 patients with chronic liver diseases and established the individual prediction model (IPM) according to their risk factors using logistic regression analysis. The IPM was calculated by the formula as follows: Risk index for HCC = e A , A = -6.254 + (1.729´liver cirrhosis) + (1.315´Age>40 years) + (1.263´C viral hepatitis) + (0.826´AFP>20IU/mL) + (0.775´B viral hepatitis) + (0.734´chronic hepatitis) + (0.584´heavy alcoholics) + (0.3´man) + (0.283´ALT>40IU/L) + (0.222´unknown alcoholic history). Moreover, the usefulness of the IPM was further confirmed by another prospective study in a total of 833 patients for 2 years. Three risk groups (low, intermediate and high) by the IPM excellently correlated to the development of HCC.

The application of the IPM to screening program for early detection of HCC may improve a cost effectiveness and efficacy of screening program by focusing high-risk group.

CRAIG N SHAPIRO 1 , XIAOJUN WANG 2 , XIAOFENG LIANG 2 1

Hepatitis B vaccine has been recommended in China as a routine infant immunization since 1992. However, until recently, the vaccine has not been provided free of charge to families of infants. This has resulted in uneven implementation of the program, with lower hepatitis B vaccine coverage in remote areas and in areas of low socioeconomic status. A national coverage survey in 1999 showed that overall hepatitis B vaccine coverage among two-year-old children was 70%, but substantially lower in the western provinces. Since then, several projects have focused on improving hepatitis B vaccine coverage and timeliness of the hepatitis B vaccine birth dose, and a regulation was issued in late 2001 by the Ministry of Health and the Ministry of Finance calling for integration of hepatitis B vaccine into the routine EPI program. The largest of these projects is the Ministry of Health/ Global Alliance for Vaccines and Immunization Hepatitis B Project, which is a 5 year, $US 76 million project to provide hepatitis B vaccine and injection equipment for newborns in the 12 western provinces and officially designated poverty counties in the rest of China. Preliminary results have shown that immunization efforts since 1999 have substantially increased hepatitis B vaccine coverage, birth dose timeliness and immunization injection safety in China.

Most patients with hepatocellular carcinoma (HCC) are not candidates for resection because of tumor size, location, multifocality, or liver dysfunction associated with cirrhosis and chronic inflammation. Radiofrequency ablation (RFA) offers an alternative treatment in some unresectable HCC patients with disease confined to the liver. We prospectively evaluated the disease-free and overall survival rates in patients with unresectable HCC treated with RFA.

All patients with unresectable HCC treated with RFA between September 1, 1997 and July 30, 2002 were enrolled in a multicenter, prospective study. Patients were treated with RFA using a percutaneous or open intraoperative approach with ultrasound guidance and were evaluated at regular intervals to determine disease recurrence and survival. 

Objective To evaluate whether the long-term results after hepatectomy for large hepatocellular carcinoma (HCC) have improved over the past decade in mainland, China.

The clinical significance of hepatectamy for large HCC is still controversial.

Methods 2102 patients who underwent hepatectomy for large HCC from January 1985 to December 2001 were prospectively collected. All patients were included in the analysis and categorized according to two time periods: after 1995 (Group A) and before 1995 (Group B). Clinicopathologic data between two groups were compared. Factors associated with long-term prognosis were further analyzed.

The overall survival results were significantly better in Group A than Group B. The cumulative 1-, 3-, and 5-year overall survival rates were 71.2%. 58.8%, and 38.7% for Group A and 67.8%, 50.7%, and 27.9% for Group A, respectively. Mean period until HCC recurrence in Group A (19 months) was longer than that in Group B (16 months), but without significant difference. There was significantly difference in the survival after recurrence between Group A and Group B (median 17 vs. 10 months). The disease-free survival results after excluding hospital deaths were also improved in Group A compared with Group B, although without significant difference in statistics. The cumulative 1-, 3-, and 5-year disease-free survival rates were 61.5%, 38.6%, and 23.8% for Group A and 56.5%, 34.7%, and 18.9% for Group B, respectively.

Hepatic resection in carefully selected patients with large HCC can be performed safely, and both overall and disease-free survival results has been improved over the past decade. An aggressive treatment of recurrent tumors using repeat hepatic resection and RFA combined with or without TACE can significantly improve the long-term survival after resection of large HCC.

Hepatocellular carcinoma (HCC) patients with the same pathologic tumor-node-metastasis (pTNM) stage of disease can have remarkable differences in disease outcome. To improve prognostication, we evaluated the gene expression profiles of HCCs to identify genes that can accurately predict disease outcome.

Gene expression profiles from 48 patients undergoing curative partial hepatectomy for HCC were included for patient outcome analysis. The importance of genes was evaluated by Cox regression and Kaplan-Meier analyses.

A set of 26 genes was identified to be associated strongly with disease recurrence after hepatectomy. An optimal gene set for prognosis was derived by including 12 top-ranked genes determined by the step-down approach, and was validated using an independent HCC sample set. A prognostic gene score for each patient was generated based on the proportion of genes that demonstrated expression level associated with poor prognosis. The prognostic gene score was 97.8% and 89.3% accurate in predicting HCC recurrence and death, respectively, within 3 years. A poor prognostic gene score was a strong predictor for recurrence and death (relative risk of 57.7 and 16.9-fold, respectively) compared to the pTNM system (relative risk of 2.4 and 5.4-fold, respectively). The prognostication by gene expression profile was independent of pTNM stages by multivariate analysis.

The gene expression pattern can provide accurate prognostication compared to the conventional systems based on clinical and pathological parameters. It can help selecting patients with poor prognosis for aggressive adjuvant therapy, and the prognostic genes may serve as therapeutic targets for disease treatment.

Angiogenesis is fundamental to tumor growth and metastasis. Hepatocellular carcinoma (HCC) is a hypervascular tumor characterized by neovascularization. Angiogenesis provides a target for novel prognostic and therapeutic approaches to HCC. Assessment of microvessel density using immunohistochemical staining for specific endothelial cell markers has been shown to provide prognostic information independent of conventional pathological parameters in HCC patients. Assessment of expression of angiogenic factors like vascular endothelial growth factor (VEGF) is a more objective way of evaluating angiogenic activity of HCC compared with microvessel density. Tumor expression of VEGF has been shown to correlate with tumor invasiveness and intrahepatic metastasis. Other angiogenic factors such as basic fibroblast growth factor and angiopoietin are also involved in angiogenesis in HCC. As most angiogenic factors are soluble factors, it is possible to measure the circulating angiogenic factors as an indirect assessment of tumor angiogenesis. Recent studies have led to the discovery of new angiogenic factors in HCC, such as tissue factor and SPARC, which may serve as new target for antiangiogenic therapy.

Studies in animal models have demonstrated the efficacy of antiangiogenic agents in suppressing hepatocarcinogenesis and growth of HCC. Antiangiogenic therapy using approaches such as anti-VEGF has already entered clinical trials in HCC patients and holds the promise of providing an effective novel treatment for HCC, which is of great clinical significance because there is no existing effective systemic therapy for HCC.

Hepatocellular carcinoma (HCC) is one of the major malignant disease in the world and ranks fifth in overall frequency. It is believed that chronic hepatitis B virus (HBV) infection is a major global cause of HCC, and HBx protein is one of the oncogenic viral proteins of HBV and is essential for natural viral infection and replication.

We have constructed a conditional HBx-expressing hepatocyte cell line, X18, by transfecting HBX into AML12 cell line, which has the unique characteristic of being non-tumorigenic while possessing a large complement of hepatocyte markers.

Gene expression profiles of the X18 cells induced in HBX expression were compared with that of the non-induced X18 cells by microarray experiments, upon the induction of HBx, 276 spots showed consistent up-regulation and 244 spots showed consistent down-regulation. At the protein level, 2D gel electrophioresis and mass spectroscopic analyses were also used to study the differential protein expression pattern upon the induction of HBx. One of the down-regulated protein spots was identified as mouse Keratin 8 (K8). However, K8 was not found to be one of the down-regulated candidate genes in microarray experiment. Thus decreased expression of K8 protein could be due to changes of its post-translational modification. Our results demonstrated that HBx might not only affect the transcription levels of its target genes, but also affect the expression of the cellular targets at the level of translation or post-translational modifications. Other genes induced in response to induction of HBx include genes of the MAPK pathway, consistent with studies reported by others 1 .

Signal regulatory proteins (SIRPs), which are also called BIT, SHPS-1 or MyD-1, are members of Ig-SFs characterized by three homologous extracellular Ig-SF domains, different transmembrane and dispensable intracellular regions. One subset of SIRPs, called SIRPa1, contains cytoplasmic sequences characteristic of SIRP proteins with no full length homologous sequences in molecular sequence databanks. In the intracellular part of SIRPa1, there are four potential immunoreceptor tyrosine-based inhibitory motifs (ITIM) which is the landmark sequence feature of a group of proteins named coinhibitory receptor and SIRPs were dubbed as inhibitory receptor accordingly. Experimental data showed SIRPa1 functions in mechanisms similar to inhibitory members of other coinhibitory receptors to inhibit cellular signaling pathways induced by molecules such as EGF, PDGF, insulin and growth hormone. Tyrosines embedded in ITIMs can be phosphorylated by various tyrosine protein kinases, and the phosphorylated tyrosines endow the hosting molecules the ability of Src-homology 2-domain (SH2) binding. The binding of SH2 domains of SH2-containing phosphatases (SHPs) like the SHP-1, SHP-2 and the inositol phosphatase SHIP in turn dephosphorylates specific protein substrates involved in mediating various physiological effects.

In hepatocellular carcinoma (HCC), SIRPa1 expression levels were seemed to be downregulated in more than 50% of HCC tissues, indicating a negative regulatory role of SIRPa1 in this disease progression and invasiveness. Exogenous expression of wild-type SIRPa1, but not of a mutant SIRPa1 lacking the tyrosine phosphorylation sites, in SIRPa1 negative Huh7 human HCC cells resulted in suppression of tumor cell growth both in vitro and in vivo. Treatment of Huh7 transfectants with EGF or HGF induced tyrosine phosphorylation of SIRPa1 and its association with SHP-2, which were accompanied by reduced ERK1 activation. Expression of SIRPa1 exerted no evident influence on TNFa/Cisplatin-induced JNK and p38 activities but significantly suppressed activation of NF-kB thus sensitizing Huh7 cells to TNF or cisplatin-induced cell death. In addition, SIRPa1transfected Huh7 cells displayed reduced cell migration and cell spreading in a fashion that was dependent on SIRPa1/SHP-2 complex formation. In conclusion, these results suggest that SIRPa1 functioned as a tumor suppressor in hepatocarcinogenesis, at least in part, through inhibition of ERK and NF-kB pathway.

To further investigate the role of SIRPa1 on hepatocyte proliferation, we established a rat liver regeneration model through 70% PH to assess the possible role of SIRPa1 involved in this process. Adult male Sprague-Dawley rats were underwent approximately 70% partial hepatectomy (PH) or sham operation (SO). Liver specimens were collected at 2, 6, 12, 24, 30, 48, 72, 120, 168 and 240 h after PH or SO. SIRPa1 expression was determined in mRNA level by Northern blotting as well as in protein level via immunohistochemical staining. SO treatment did not induce remarkable changes in SIRPa1 expression, however, the level of a 3.9kb transcript for SIRPa1 was significantly upregulated after PH (vs SO, p<0.05). SIRPa1 mRNA level in the regenerating liver displayed a biphasic response with its first large peak at as early as 12 hours followed by a second phase of upregulation from 48 to 120 hours post-PH. 168 hours later, SIRPa1 mRNA level returned to its physiological level. As seen from immunohistochemistry experiment, SIRPa1 protein mainly located in membrane and was expressed uniquely in regenerating hepatocytes. Similarly, PH-induced overexpression for SIRPa1 protein occurred between 12 and 168 hours with a peak level at 24 hours after surgery. Taken together, these data strongly suggest that SIRPa1 may play an important role in controlling early hepatocytes proliferating cycles. Unlike those early response genes such as c-fos and c-myc, SIRPa1 may be involved in the regulation of liver regeneration in a negative fashion. As we known, liver regeneration is regulated by a perfect cooperation of various proteins, including positive and negative regulators that are almost simultaneously activated, for a well-tuned control on proliferative initiation and termination. To date, liver regeneration has been largely focused on the positive regulators while little is known for the negative regulators. As negative regulator, SIRPa1 may cooperate with other specific candidates in terminating the proliferative signals to prevent excessive proliferation of liver cells.

In summary, SIRPa1 may play negative regulatory roles in HCC development and progression as well as liver regeneration in response to PH. Further studies are underway to disclose the precise mechanisms governing the phenomena.

The structure of cell surface glycans is closely related to the malignant behaviors of cancer cells, including metastasis. In our laboratory, it was found that the b1,6 GlcNAc (N-acetylglucosamine) branching structure on Asn-linked glycans (N-glycans) and the sialylated fucose (Fuc)-containing Lewis X sugar antigen [SLe x , SAa2,3 Galb1,4 (Fuc a1,3)GlcNAc-] mainly located at the terminal of Ser/Thr-linked glycans (O-glycans) and glycolipids are two of the important metastasis-associated glycan structures for human hepatocellular carcinoma cell line, H7721. Beta-1,6 GlcNAc branch is synthesized by N-acetylglucosaminyltransferase V (GnT-V), and the enzyme for SLe x synthesis is a1,3 fucosyltransferase (a1,3FucT) family, including subtype-VII VI and-III. 

Hepatitis B virus is one of most common pathogens in the world. Current estimates are that 2 billion people have been infected worldwide[1,5,13], 360 million suffer from chronic HBV infection resulting in over 520 000 deaths each year. In China, there are an estimated 120 million hepatitis B carriers, defined as persons positive for hepatitis B surface antigen (HBsAg) for more than 6 months. About 28 million patients are currently suffered with chronic hepatitis B, The strategy of Anti-viral therapies and vaccination were taken into consideration and widely accepted in China. HBV virological diagnosis is an important part of Chinese Chronic Hepatitis B Practice Guideline. Now the development of diagnostic tests continued and the qualities of laboratory tests were improved.

The virological diagnosis of HBV combined with serologic assays and molecular methods. Currently serologic assays served as basic test of hepatitis B virus, however, the molecular methods were widely used in clarification of chronic hepatitis B and evaluating the response of therapy as well as laboratory researches.

In China, there are three systems, HBsAg-Anti-HBs, HBeAg-Anti HBe, total and IgM anti-HBc respectively, used frequently.

HBsAg is glycoprotein expressed on the surface of the HBV virons, encoded by S, pre S1 and pre S2. The positive result of HBsAg may suggest the early period of HBV infection, The occurrence ranged from 5 weeks to 5 months after infection. If the persistence of HBsAg positive exceed more than 6 months, It suggest that this patient is with chronic hepatitis B virus infection. Anti-HBs is the antibody produced by host against the HBsAg, named as protective antibody. It occurred after acute HBV infection and produced in resolved period accompanying with specific immune response, also occurred in the resolved chronic hepatitis B patient, it can be used to monitor the effectiveness of HBV vaccine.

According to the HBsAg, HBV have been divided into four serological subgroups, adw, adr, ayw, ayr respectively. In china the predominant types are adr and adw.

HBeAg is derived from the translation product of the precore and core region of HBV genome. it is secreted from the infected cell but is not part of the virion. In the acute infection, the occurrence of eAg is later than the HBsAg [7], but it becomes undetectable before HBsAg is cleared. In chronic hepatitis B, HBeAg concentrations parallel concentration changes in HBV DNA. Persistence of HBeAg for more than 12 weeks indicates chronicity. HBeAg has been used as a marker if viral replication and as an index that correlates with infectiousness of the patient and severity of disease. Anti-HBe is antibody produced by immune response against HBeAg, The first detection of anti-HBe may actually precede loss of HBeAg but usually there is a small lag time between two markers. The occurrence of Anti-HBe means that the replication of HBV is decreased. The disappearance of HBeAg and the occurrence of Anti-HBe were defined as seroconversion. Seroconversion is usually used as a marker to monitor the response of Interferon-and nucleotide analogue treatment.

The viral core of HBV is composed of nucleic acid, a DNA polymerase, and an antigenic nucleoprotein. Anti-HBc is perhaps the most serologically prominent marker of HBV exposure. HBcAg is very immunogenic, and consequently high anti-HBc titers early in an infection suggest a period of active viral replication. Tests for IgM anti-HBc are used for the differential diagnosis of acute disease. The earliest anti-HBc in acute disease is predominantly IgM antibody, with low activities of IgG anti-HBc also present concurrently. The titers and percentage of IgG anti-HBc continued to rise during early convalescence. In chronic hepatitis B patients, IgG anti-HBc presents with high concentration but the IgM presents with lower titer [12] .

In China, Detect assays compounds of EIA, RIA and MEIA, provided by Abbott, Roche, Qiansen and Kehua Company.

Nucleotide assays can be used to detect HBV-DNA, HBV genotype and variant or mutation analysis[8]. HBV DNA: HBV has a small (3.2kb) circular DNA that is mostly double stranded but contains a short region of single strand [6]. The positive result of HBV DNA detection confirmed the currently infection, as a marker of viral replication during acute and chronic hepatitis B. Quantitative HBV DNA test used widely to monitor the dynamic of viral load variation during antiviral treatment. With the data of HBV DNA variation during therapy, the response can be defined as: 1).End-of-treatment response, 2). Sustained response, 3). Breakthrough and Relapse. The HBV DNA level before treatment can predict the efficiency of antiviral therapy. In recent years, the early virologic response has been taken more attentions, the slope of HBV DNA decreasing in first period means the clearance of virus in the blood, and the slope of HBV DNA decreasing of the second period means the clearance of the virus in the hepatocytes [1, 2] .

The results of HBV DNA detection can be expressed qualitatively and quantitatively [8] . The tests are of three types: direct hybridization, branched chain (b-DNA), and polymerase chain reaction methods. In China, the commercial available test included b-DNA, Digen, Amplicor and COBAS Amplicor, the most extensive method is Real-time PCR. The excellence of hybridization is the high specificity but low sensitivity, but the assays based on PCR have good sensitivity but several factors can influence the results. The lower limit of detection of these assays arranges from 102 to 106 copies/ml. The figure followed described the range of detection.

The genotypes of HBV may be differentiated on the basis of HBV genetic sequence or S epitope variation, currently there are seven genotypes (A-G), the distribution of HBV genotype is differentially in the world. In China, Genotype B and Genotype C are the main genotype. Currently the genotypes were determined by sequence, RFLP or gene-chip assays in china. The correlation between genotypes and efficiency of antiviral therapy were still on study in China.

HBV variant is characterized by any naturally occurring variation from published wild-type sequences. In HBeAg-negative chronic hepatitis B, A single change of one base-pair at position 1896 of this region is sufficient to account for this, because the change converts the triplet codon at the site into a "stop" codon. Therefore, during synthesis of the viral proteins the precursor of HBeAg is not produced. Once this variant of HBV become the main strains and accompany with the BCP mutation, the severity of liver diseases may be enhanced.

A HBV mutant is defined as a variant that develops under specific selection pressure and that has been shown to confer a specific phenotype. Marked fluctuation in hepatitis activity and viral replication are common, Because of the need for long-term treatment, therapy is indicated only for patients with moderate to severe chronic hepatitis. The relapse occurred common in this group after treatment. In clinic practice, diagnostic criteria is the positive result of HBV-DNA coupled with HBeAg negative /anti-HBepositive. In China, about 20-40% of chronic hepatitis B patients are anti-HBe positive CHB patients, the rate of the anti-HBe positive CHB seems to increase in China. The diagnosis of anti-HBe positive CHB is based on sequence, RFLP and genechip.

S escape mutant have been reported, the vaccine failed to protect the vaccinated children, and the liver transplant patients developed the S escape mutant during HBIG treatment. Now Abbott's kit can detect the HBV S escape mutant [4] .

Use of nucleotide analogue has been complicated by the emergence of antiviral drug resistance mutants as a direct consequence of changes in the polymerase gene. Because the polymerase is the target of NA treatment, the drug-resistance mutant was selected under the pressure of the nucleotide analogue [9,10]. The commonest polymerase gene mutation associated with resistance to lamivudine treatment is the methionine to isoleucine amino acid substitution at position 204 of the viral polymerase(rtM204I). The patients with YMDD mutant can have elevated ALT and lead to exacerbation. Several assays can detect YMDD mutant, such as RFLP, Realtime PCR and line probe. During the treatment, the patients can be monitored by HBV-DNA, ALT and YMDD detection.

The serologic test had a great development and became popularization in the past 20 years. In recent years, nucleotides test developed very quickly, but the distance between different regions and different hospitals still very large. The problems need to be resolved in the future as describing below:

1. Improve the sensitivity and specificity of serologic detect kit homemade, develop the quantitative test of anti-HBs, HBeAg and anti-HBc-IgM.

With the peglated Interferon [14] and number of nucleotide analogues used in China, the quality of quantitative test of HBV DNA and detection of drug-resistance mutant need to be monitored more intensively.

Study further the potential markers [3], which can suggest the process of disease and predict the response of antiviral therapy [11], such as cccDNA, specific cell immune response.

Apoptosis is one of the fundamental directions of cells, in which the cell death is operated by a series of genes functioning in triggering, transducing, regulating, and executing the death signal. The aberrant Fas or TNFa mediated apoptotic signaling has been implicated in the pathogenesis of many types of liver diseases including fulminant hepatitis, acute and chronic viral hepatitis, autoimmune hepatitis, cholestatic liver injury, and alcohol, chemical or drug induced liver intoxication. Bid is believed to be an intracellular pro-apoptotic mediator of the signaling from both Fas and TNFR1 activation in liver. Therefore, protection of liver from excessive cell death caused injury represents a new strategy to treat certain types of hepatitis. Antisense technology has been broadly used in specific suppression of gene function at RNA levels both in vitro and in vivo. The first antisense drug for treatment of cytomegalovirus (CMV) retinitis in people with AIDS was approved in 1998 by FDA. A number of antisense compounds are progressing in clinical trials for treatment of various diseases including liver relative diseases (HCV infection, diabetes, lipoprotein metabolism, etc.). To explore the therapeutic application of anti-apoptotic injury in liver, we use antisense oligonucleotides to inhibit a number of apoptotic genes expressed in liver and examine their pharmacological effects in hepatitis models. 

The antisense oligonucleotides suppressed the expression of targeted genes in mouse liver after intraperitoneal administration in a saline solution. The reduction of target expression was antisense sequence specific, target sequence specific and antisense dose dependent. Treatment with Fas antisense significantly reduce serum ALT and apoptotic injury in both Fas antibody and acetaminophen induced liver injury and completely protected mice from fulminant death induced by Fas activation. Treatment with TNFR1 antisense protected mice from liver injury and death in LPS/D-galactosamine induced, but not Fas antibody induced, fulminant hepatitis. Treatment with Bid antisense protected mice from the fulminant death in both Fas antibody and LPS/D-galactosamine models. In addition, treatment with Bid antisense markedly reduced hepatocyte apoptosis following exposure to bile acid (glychochenodeoxycholate, GCDC) and significantly attenuated liver apoptosis and serum ALT increase in the cholestatic hepatitis by bile duct ligation.

These results suggest that protection of liver from apoptotic injury is a promising approach for the therapy of fulminant hepatitis, cholestatic hepatitis, and other hepatitis that involves apoptotic injury in the onset. Antisense oligonucleotides with 2'-O- (2-methoxy) ethyl modification are demonstrated to be effective pharmaceutic compounds by suppression of aberrant gene functions in relation to the pathogenesis in liver for a variety of liver related diseases.

Many determinants for a sustained response to lamivudine thterapy have been reported but the role of T cell responsiveness remains unclear. The finding that tyrosine-methionine-aspartate-aspartate (YMDD) motif of the reverse transcriptase domain of HBV DNA polymerase carries an HLA-A2-restricted cytotoxic T lymphocyte (CTL) epitope makes a quantitative measurement of the numbers of peptide-specific CTLs feasible by MHC tetramer-peptide complexes staining.

To investigate the correlation of anti-YMDD motif CTL activity with the efficacy of lamivudine therapy in HLA-A2 positive patients with chronic hepatitis B (CH-B).

The function and phenotype of peptide-and IL2-expanded PBMCs were quantified by cell lytic assay and immunocytochemistric analysis by staining with HLA-A2 -peptide tetramer complexes .

After in vitro expansion, the sustained responders possessed more potent CTL responses against YMDD, YVDD and YIDD as well as other epitopes on HBV antigens than the non-responders. The frequency of YMDD/YVDD/YIDD motif-specific CTLs increased significantly with an effective cell lytic function during and after therapy in sustained responders but not in nonresponders. YMDD-specific CTLs cross-reacted against YIDD-and YVDD mutant epitopes, and shared T-cell receptor gene usages with YIDD-and YVDD-specific CTLs.

Sustained responders, at least HLA-A2 patients, elicited a more potent CTL immunity against YMDD and its mutants. YMDDspecific CTLs are cross-reactive with YVDD and YIDD mutant epitopes, which may further contribute to immune clearance of the mutant viruses, and a successful response to lamivudine therapy in CH-B patients.

The dogma for tumor evolution is that a tumor is derived from a clonal expansion of an initiating cell with a mutation in a tumor suppressor gene or oncogene, followed by an acquisition of sequential multiple genetic changes. A working template was established with the hereditary colorectal cancer model over a decade ago by Fearon and Vogelstein. This model also indicates that the primary tumor should start off benign but, over time, acquire mutations that provide a few rare cells within the tumor the ability to metastasize. Likewise, the development of hepatocellular carcinoma (HCC) is expected to follow a multi-step process. We have been using DNA microarray and SAGE to define molecular signatures during HCC progression. Our strategy is to identify cellular genes that are commonly changed by the expression of HBV or HCV in primary human hepatocytes, preneoplastic chronic liver disease and HCC. By comparing cirrhotic liver samples from chronic liver disease patients from various etiological factors, we have identified a molecular signature that may be useful in diagnosing patients at risk for developing HCC. By comparing primary HCC with or without accompanying metastasis, we have identified a molecular signature that can identify HCC patients with potential in developing metastasis or recurrence.

Using gene expression profiling, we have obtained evidence suggesting that at least some types of HCC may not follow the conventional clonal selection and expansion model. First, we found that a significant amount of cancer-associated genes were differentially expressed in various parts of premalignant liver tissues without avert HCC from chronic liver disease patients who had a high risk of developing HCC. This data suggests that a high rate of malignant changes took place in premalignant tissues prior to clonal expansion. Second, we found that the gene expression signature of primary HCCs with accompanying metastasis was very similar to that of their corresponding metastases, while the molecular signature differs significantly in primary HCC with or without metastasis. These results indicate that most of the metastasis genes are embedded in the primary tumors and that the ability to metastasize is an inherent quality of the tumor from the beginning. The above findings challenge our current view on HCC progression. Using the above approaches, we have identified several diagnostic markers and potential therapeutic targets that can eliminate liver cancer cells or stop metastatic progression. These studies also provide us with a strategy that may be useful in the future to tailor HCC patients, based on their gene expression profiles, to adjuvant therapy. Currently, we are exploring the roles of these genes in liver cancer initiation and tumor progression.

Etiologically, the epigenetic disturbance has been implicated in human cancers, where both global demethylation and focal hypermethylation of the promoter CpG islands occur prevalently. The former is at least partly responsible for loss of the genome stability at both gross and fine levels and the later has been implicated as the key alternative to the genetic alteration for the inactivation of the tumor suppressor gene in tumors. The hepatocellular carcinoma (HCC) is one of the most threatening cancerous diseases in China, for both its high incidences and the difficulties in early diagnosis and the poor prognosis by the existing treatments. To understand the epigenetic mechanisms ( more specifically, DNA methylation) in HCC carcinogenesis and develop the novel robust diagnostic, prognostic and therapeutic tools, we have methylation-profiled forty four known genes in twenty eight HCC tissues, the neighboring normal tissues as well as four normal liver tissues from the healthy donors. The same efforts have been devoted to the astrocytoma, colon and lung cancers. We concluded from such the rather intensive methylation profiling survey: 1, the tumor associated epigenetic aberrations is global, would also affect the genes with no demonstrated roles in carcinogenesis. 2, the correlation between the changes in the methylation patterns and of the clinicalpathological parameters (cirrhosis in particular) has be recognized and may have certain clinical implications. 3, the MAGE1A gene is hypermethylated and lacking of expression in the normal liver and hypomethylated and over-expressed in HCC at the high frequency, probably representing the first case of the tumor associated hypomethylated promoter CpG island in connection with the gene expression. And 4, the concordant behavior of methylation among all the informative targets can be extrapolated with the necessary mathematic treatments and can be used for the target selection in the future DNA methylation based prognostic and diagnostic tools for the human tumors. We are currently looking for the novel associated genes with the HCC specifically altered DNA methylation status by theomics approaches.

Finally, to unveil the epigenetic control mechanisms in gene expression, we are using the MAGE1A gene as the model system to systematically assess the contributions of the DNA methylation, histone modification and chromatin remodeling to the transcription of this gene in the context of the carcinogenesis of hepatocellular carcinoma.

A variety of approaches including purification of subunit of HBsAg from plasma and expression of HBsAg by genetic engineering have been applied in development of vaccines against HBV infection. Recently, increasing evidences indicated that DNA vaccination, a recently established novel immunological theory and practice, has been proposed as a new, simple and cost effective way to evoke immune responses to HBV. However, its efficiency in induction of both humoral and cellular immune response remains to be raised. Theoretically, the efficiency of DNA vaccine could be improved by increasing transduction in vivo, heightening production and possibly secretion of the transgenic product, and enhancing antigen presentation. From a practical standpoint, in this study we focused our efforts on attracting antigen presenting cells to the site of antigen existed, increasing expression of transgenic product, enhancing antigen presentation and activating APC.

Chemoattraction of APC to the site of antigen existed RANTES, a chemokine considered to be able to attract professional APC was introduced into HBV DNA vaccine. Intramuscular injection of a plasmid DNA containing RANTES coding sequences led to infiltration of inflammatory cells including macrophages and dendritic cells in the site of DNA inoculation. Co-injection of pRANTES with a plasmid DNA harboring hepatitis B virus envelope gene (pHBs) resulted in enhancement of viral specific immunity comparing with that induced by pHBs only, indicating that attraction and recruitment of more APCs to the site of antigen existed by chemokine might be a way to improve the efficiency of DNA vaccine.

T7 promoter is one of the most powerful transcriptional regulatory elements for the expression of foreign proteins in vitro. However, it can be only used in prokaryotic system. Introduction of T7 promoter into eukaryotic system could increase transgenic production and significantly elevate the immune response consequently. In our study, two plasmids were constructed: pCMV-T7 in which T7 RNA polymerase encoding gene was under the control of eukaryotic CMV IE promoter, and pT7-HBs in which a gene coding for hepatitis B virus envelope protein was cloned downstream of prokaryotic T7 promoter. Co-immunization of BALB/c mice with PCMV-T7 and pT7-HBs resulted in a higher expression level of HBsAg and sequentially a stronger humoral immune response, suggesting that eukaryoticalization of prokaryotic expression system is an alternative way to enhance immunity induced by DNA vaccine.

It is well known that epitopic peptides generated by processing in the cytoplasm are selectively transported into the endoplasmic reticulum (ER) in which they bind to newly synthesized MHC class I molecules. Therefore, measures aimed to guiding epitopes into ER could possibly enhance antigen presentation via MHC class I pathway. P18, a fragment derived from Adenovirus E3 leader sequence with ER insertion signal sequence (ERISS) was hypothesized to facilitate transport of epitopes into ER for binding to MHC class I molecules. To this end, a CTL epitope (C 18-27 ) coding gene derived from HBV was fused with P18 (pEC K -E3-C 18 ) and then intramuscularly inoculated into C57BL/6 mice. Measurement on CTL responses specific to HBV indicated that CTL activity was significantly enhanced in mice immunized with the construct harboring both P18 and CTL epitope sequence.

Our previous studies have demonstrated that P28, derived from the C3d component of complement, can specifically bind to CR2 receptors on B cells. In this study, the potential adjuvant effect of P28 peptide for enhancing anti-HBs immune responses was evaluated using a DNA vaccine. Plasmid encoding a fusion protein of preS2/S of HBV and P28 of C3d was constructed. Analyses of the antibody titer and avidity maturation of the raised antibody indicated that immunization with the plasmid DNA induced higher primary humoral responses as well as a faster and stronger memory reaction, which correlated with accelerated avidity maturation of anti-HBs antibody. This accelerated avidity maturation of antibodies against HBsAg might also correlated with the higher expression of CD40L, IFN-g and IL-4 from isolated splenocytes, indicating the adjuvant effect of P28 for enhancement of the immune responses to a conjugated antigen.

Transarterial chemoembolization (TACE) involves the delivery of chemotherapeutic agent(s) combined with embolization of the hepatic artery to destroy liver cancer.

Restrospective and non-randomized studies have shown TACE to give good results. Five randomized control trials (RCT) have compared TACE with supportive treatment, with 3 showing TACE to have no impact on patient survival. Two RCTs showed TACE to be effective in prolonging the lives of patients with unresectable HCC. These 2 studies also emphasize on the selection of patients with less advanced HCC before the patients can benefit from the treatment.

TACE has been shown to be able to downstage unresectable HCC to become resectable, thus providing a possible cure to patients with advanced HCC.

Retrospective studies suggest that neoadjuvant TACE is safe and feasible. However, the results of RCTs are controversial. Two systematic reviews concluded that there is no evidence on the use of neoadjuvant TACE in HCC.

Adjuvant TACE after curative resection produces controversial results in non-randomized and randomized studies. Two systematic reviews concluded that TACE has not been useful.

The use of neoadjuvant / adjuvant TACE has not been tested within the context of RCT.

TACE produces worse results than re-resection if the tumours are still resectable. It is uncertain whether TACE is better or worse than percutaneous ethanol injection (PEI) or radiofrequency ablation (RFA).

Non-randomized studies showed that TACE and PEI produced good results. However, two RCTs showed controversial results as to whether TACE + PEI were better than TACE alone.

Preliminary data showed promising results with TACE combining with RFA, debulking surgery, cryosurgery and radiotherapy.

Other Procedures Aiming at "Cure" for HCC TACE has been combined with PEI in a RCT to produce superior long-term survival when compared with PEI alone.

TACE is becoming established in treating unresectable HCC. Whether it is an effective bridge to liver transplantation remains to be determined. Whether TACE should be combined with another procedure in the curative or palliative treatment of HCC is still uncertain. 

Primary liver cancer is one of the most common malignancies in China. Although liver resection is the optimal strategy for liver cancer, low resectability and high postoperative recurrence rate remain to be the main barriers for favorable long-term survival. Therefore, comprehensive treatments for PLC, which aims at improving the overall therapeutic efficacy, has been the focus of clinical work and research: (1) The volume of liver removal: as anatomical or extended hepatectomy may result in severe decompensation of liver function, modality of liver resection has been shifted from extended resection to irregular radical local resection. Substantial clinical practices have shown that in patients with liver cancer associated with chronic hepatitis or cirrhosis, radical local resection not only increased resectability but significantly decreased operative mortality and achieved long-term outcomes similar to, or even better than those achieved by extended resection.

(2) Complications with portal vein or biliary tract cancerous thrombi: hepatectomy plus removal of cancerous thrombi as well as postoperative chemotherapy may prolong the survival time and improve the quality of life for selective liver cancer with portal vein or biliary tract cancerous thrombi. The 2year survival rate was up to 36.6%, higher significantly than that of non-surgical interventions. As a result, we advocate that positive operation is worthwhile only if tumor is resectable for those patients. Especially obstructive jaundice due to cancerous thrombotic invasion of the biliary tract may disappear gradually.

(3) Complication with portal hypertension : in patients with splenomegaly, hypersplenism and hemorrhagic or non-hemorrhagic esophageal varices, (where use of portocaval shunt is quite rare), hepatectomy plus splenectomy and occlusion of the varix have been attempted. Combination of these two or three procedures has resulted in outcomes similar to hepatectomy in patients without portal hypertension. In our studies, the 2-and 3-year survival rates were 73.1 percent and 47.5 percent respectively. (4) Two-stage resection: comprehensive therapies for shrinking liver tumors available at present include TACE, PEI, RFA and cryotherapy, etc. Rational use of these techniques is crucial for tumor shrinkage. Two-stage resection is indicative for those definitely unresectable tumors, and otherwise one-stage removal is still recommended to be the optimal choice in the management of liver cancer. (6) Prevention and treatments for postoperative recurrence of liver cancer: intraoperative preventive measures, postoperative comprehensive therapies against recurrence, and early detection and management of possible recurrent lesions are important strategies to improve the therapeutic effects. Fine surgical manipulation to avoid medical dissemination, non-touch technique, suction of cancerous thrombi and portal chemotherapy are essential for prevention of recurrence. Rational use of the comprehensive therapies according to the specific conditions of individual patients is of critical importance. B-US, AFP and chest X-ray should be performed regularly.

Clinical practices have shown that DSA one month after operation can early detect residual cancerous lesions and micro-lesions of intrahepatic metastasis so as to save precious time for further therapeutic attempts. Reoperation is an effective strategy for the improvement of 5-year survival rate for both recurrent tumors amendable to surgical operation. In our data, the 5-year survival rates after a second and third operation are 46.6 percent and 25 percent respectively. (7) Liver transplantation: previous series concerning OLT in liver cancer demonstrated a poor long-term survival because most recipients selected for OLT were patients with terminal stage of unresectable tumors and immunosuppression treatment was liable to accelerate growth of the liver cancer or promote tumor recurrence. For small liver cancers, transplants eliminate both detectable and undetectable tumor nodes and all preneoplastic lesions in the cirrhotic liver. Removal of the diseased liver also reduces the risk of morbidity and mortality from portal hypertension. At present, liver cancer is still an indication of liver transplantation, where relatively better long-term survival can be expected for early stage liver cancer, incidental cancer and fibrolamellar hepatocellular carcinoma. OLT is selective for patients with tumors less than 5cm in diameter that show no vascular invasion, decompensated liver cirrhosis or contraindications for resection. Satisfactory OLT outcomes may be expected in stage II or III liver cancers providing that perioperative treatment is meticulous enough. OLT is contraindicated for late stage liver cancer. (8) Non-surgical interventions: on patients with liver cancer who can't accept operation for various reasons, TACE, PEI, or RFA may have resulted in favorable outcomes. Attentions are essentially placed on the sequences of various interventional therapies. The action of TACE depends on the tumor artery, whereas PEI , RFA and other procedures may destroy the vascular system of tumor. Therefore, TACE is essential to be preferential. In summary, comprehensive treatments has become a more important strategy for improving the overall therapeutic effect on liver cancer. Two principles should be keep in mind: (1) attention should be devoted to the complimentary effects of each method. (2) avoiding the counteraction of the effects or the accumulation of side effects.

Hepatocellular carcinoma (HCC) tends to invade the portal vein resulting in the formation of portal vein tumor thrombus (PVTT). PVTT was proved to be an important negative prognostic indicator of this disease. From January 1998 to December 2002, 917 patients with liver cancer after liver resection were enrolled in our study. Of these, 797 patients (86.9%) had PVTT if all degrees of portal vein are included and 139 patients (15.2%) had gross PVTT (PVTT in portal trunk, its first-order branches and its second-order branches). The diagnosis of gross PVTT was verified mainly by image technical such as ultrasound, CT or MRI. The existence of gross PVTT was confirmed by preoperative image in 92 cases (66.2%), intraoperative exploration in 32 cases (23.0%), and histopathological examination in 15 cases (10.8%). Although PVTT in portal trunk may be from the HCC located in different segments (34/139, 24.5%), it was mainly caused by tumor in right lobe (21/80,15.1%). Tumor usually invaded the portal vein in same side. It is rarely seen that tumor in right lobe infiltrated left portal branch (3/80, 0.04%) and tumor in left lobe protruded to the opposite-side portal branch (2/36, 0.06%). Hepatocellular carcinoma seemed to be easier to invade the portal vein than cholangiocarcinoma or mixed type, as the incidence rate of PVTT in each type was 15.86%, 4.44%, 9.52% respectively, but they had no significent difference. While the tumor size has closed relationship with PVTT incidence in this series because the incidence rates was 7.6% (£3cm), 7.72% (3~5cm), 16.4% (5~10cm) and 22.4% (e"10cm), respectively. The incidence rate showed significant difference when taking 5cm as critical point (p0.01). In this series, all patients received hepatectomy and intraoperative chemotherapy via portal vein. PVTT resection or aspiration was performed in 124 patients who had the presence of gross PVTT. However, the reapperence of gross PVTT was detected in 79% patients within 60 days after operation, with even more serious symptoms. In view of the limitations of surgery in the management of recurrent PVTT, the new therapeutic approach has been developed on the basis of minimally invasive therapy. In this study, 65 cases were randomized separated into three group treated with different modalities including LA (laser ablation, n=24), TACE (n=20) and PPVC (percutaneous portal vein chemoembolization, n=9). the regression of PVTT and the reappearance of portal vein flow detected by color Doppler Ultrasonograpy were used as evaluating criterion for the effecacy of therapy. The regression of PVTT and the continuous reappearance of blood stream in patients treated with LA, TACE, PPVC was 92.3%, 10%, 26.3% respectively 30d after therapy, and 75%, 15.38%, 35.3% respectively 90d after therapy. Our study suggested LA might be an effective new therapeutic modality for PLC with portal trunk and its main branches tumor thrombus, although its long effects remain to be further studied.

Though HBV prevailed in China by a high infection rate and causes considerable morbidity, the geographical distributions and clinical relevance of HBV genotypes in this area remain unknown. This study aimed to investigate the distribution, clinical and virological characteristics of HBV genotypes in Chinese chronic carriers.

Using a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method, we studied 1096 Chinese chronic HBV carriers from 9 provinces covered the Mainland China. All samples were genotyped by one assay in the Key Laboratory For Viral Hepatitis, Nanfang Hospital. HBV genotypes were identified by PCR-RFLP on amplicon of the small S gene as described previously.

Four major genotypes A, B, C and D were found in this study and the ratios were 1.2%, 41%, 52.5%, 4.3%, respectively. In northern China, genotype C is predominant by a ratio of 85.1%, while genotype B is more popular in the south (55%). Further analysis was carried out between the two major genotypes B and C. A younger mean age and lower positive rate of HBeAg was observed in the patients with genotype B compared those with genotype C. However, there were no significant differences in sex, levels of ALT and total bilirubin between groups B and C. Disease distributions of ASC, CH, LC and HCC in genotypes were 17.2%, 78.2%, 4.2% and 0.4% in patients with genotype B; 14.4%, 77.5%, 6.9% and 1.2% in genotype C respectively. Among 264 chronic hepatitis B patients who were performed liver biopsy in the Liver center in Nanfang Hospital, 140 were of genotype B, 124 of C. The histological indexes were compared between these two genotypes. However, there were no significant differences found either in inflammation scores (P=0.1) or in fibrosis scores (P=0.3) between the two groups. Phylogenetic analysis showed that all HBV strains of genotype B and C prevailed in China had consensus preC/C sequences.

This study illustrated the geographical distributions of HBV genotypes in Mainland China, and suggested that there may be lack of clinical differences between HBV genotype B and C, which may relate to the molecular characteristic of HBV strains. So, if stratification for HBV genotypes should be included in the clinical evaluation in this area needs further studies.

Primary biliary cirrhosis (PBC) has been recognized as an entity of liver disease since 1950s. Histopathology shows features of progressive non-suppurative destructive cholangitis that ultimately leads to liver fibrosis and cirrhosis. Although its exact pathogenesis still remains unknown, autoreactivity to intrahepatic bile duct system is believed to be the principal mechanism. In Western and Northern Europe the estimated disease prevalence varies between 20 and 240 cases per million population and an incidence between 4 and 30 cases per million per year; furthermore, the incidence of PBC appears to be rising. However, in China, PBC had been ignored and underdiagnosed for many years, probably because of the misconception that PBC was mainly involved Caucasians and the unaccessibility to specific laboratory tests. As the accessibility to world medical literature and academic exchanges have become easier and more frequent, and the wide-spread use of automatic biochemical analysis and availability of anti-mitochondrial antibody( AMA) detection, we and other Chinese hepatolgists began to realized that the PBC is much more common than we thought before. In recent years, at least more than 400 hundred cases of PBC have been reported in the mainland of China (mainly in Chinese medical literature). Actually in our own institute and one other hospital, each more than 100 hundred cases have been diagnosed in the last 5 to 10 years. As in western countries, in China PBC is predominantly affects women, with a peak age of about 50 years. The earlier series was consisted of mainly older late stage cases which had been undiagnosed until occurrence of overt jaundice, even until the development of portal hypertension and liver failure. Recent series comprised of more and more younger and earlier and even asymptomatic cases accidently identified at routine health check or workup for other medical problems. The clinical manifestation (such as fatigue, pruritus, dry mouth and eyes), biochemical( such as increased alkaline phosphatase and gamma-glutamyl transpeptidase), immunological (such as increased immunoglobulin M and AMA/AMA-M2 positivity) as well as histological (such as bile duct inflammation and destruction, dense lymphocyte infiltration in portal area and granuloma formation) features of reported Chinese cases are more or less the same as the cases reported in western countries. The biochemical response to medical therapy, namely high oral dose of ursodeoxycholic acid, was also similar to that reported in western literature, although large scale clinical trials or even retrospective series on the long-term efficacy of ursodeoxycholic acid are sill lacking. In China Orthotopic liver transplantion also has become the treatment of choice for end-stage PBC patients and not surprisingly, post-transplant survival profile was very good. Interestingly, we found some cases with typical biochemical, immunological and histological features of PBC also had markers of hepatitis B virus infecton. They showed evidences of either past( positive for anti-HBs and/or anti-HBc) or current (positive for HBsAg)HBV infection, but usually with undetectable HBV-DNA by PCR amplyfication. The causal relationship, if there is at all, between PBC and HBV infection remains to be investigated. In conclusion, because of an increased awareness of PBC among doctors and the availability of diagnostic tests, especially the measurement of AMA/AMA-M2, more and more PBC cases have been diagnosed and timely treated with internationally accepted medical and surgical modalities.

The . Those patients, 16 male and 3 female, aged 17 ~ 56 years (average 30.2 years) and had a history of HBV infection of 1~9 years (average 3.7 years). They were proved to be positive for HBsAg, HBeAg and HBV DNA (>10 5 copies/ml), without cirrhosis of liver, infection of other virus etc. Among them, two were receiving therapy of Lamivudine while others didn't have an anti-HBV therapy for at least 1 year. RESULTS: In all patients, the difference between before and after the therapy of HBV-DNA was 1.771± 2.39p<0.01;The percentage for HBeAg negative seroconversion was 52.6% (10/ 19); the percentage for HBeAg -AntiHBe seroconversion was 26.3% (5/19) . Compared with routine anti-HBV -IFN therapy, the therapy with HBsAg-pulsed DC had a better percentage for HBeAg negative seroconversion(p>0.1) ,but with a similar percentage for HBeAg -AntiHBe seroconversion(p>0.1). No safety issues of concern emerged. INVITED PAPER New insights into the antiviral mechanism of IFN-a a a a a using cDNA microarray WEI XIONG, XUN WANG, XIAOYING LIU, LI XIANG, LINGJIE ZHENG, ZHENGHONG YUAN

In the past two decades, alpha interferon (IFN-a) has been proven effective in the treatment of chronic Hepatitis B virus infections. However, the antiviral mechanism of IFN-a and the biological function of many IFN-a responsive genes have not been fully elucidated. To study the global effect of IFN-a on cellular genes and its interaction with HBV, we used cDNA microarray filters dotted with 14,000 human genes to analyze transcriptional changes between an HBV DNA transfected hepatoblastoma cell line (HepG2.2.15) and its parental cell line (HepG2) pre-and post IFN-a treatment. Analysis of gene profiles revealed that the mRNA expression level of myeloid differential primary response protein MyD88 (MyD88) was reduced in the HBV persistently infected HepG2.2.15 cells compared with HepG2 cells, and this could not be restored by the treatment with IFN-a. This result suggested that the expression of MyD88 was inhibited by HBV and the interplay between MyD88 and HBV might account for the establishment of viral persistence. This also suggested that MyD88 protein could play an important role in antiviral activity against HBV. To examine the role of MyD88 in the antiviral activity of IFN-a against hepatitis B virus (HBV), we established MyD88 stably expressing cell lines and studied HBV replication in these lines after transient transfection. The levels of HBV proteins and viral replicative intermediates were effectively reduced in MyD88 expressing cells. And a significant reduction of total and cytoplasmic viral RNAs in MyD88 stable expressing cells was also observed. Using a nuclear factor-kappaB (NF-kB) dependent reporter assay, it was shown that activation of NF-kB was moderately increased in the presence of expression of MyD88, and further significantly increased by co-expression of HBV. These results suggest a novel mechanism for the inhibition of HBV replication by IFN-a via expression of MyD88 protein involving activation of NF-kB signaling pathway and down regulation of viral transcription. Another important implication of above results is that the novel antiviral genes identified by microarrays could be potentially developed as new anti-HBV drug or for novel therapies.

Liver cirrhosis is a worldwide health problem. To develop a new therapeutic approach, we investigated the potential of recombinant adeno-associated virus (rAAV)-mediated stable heme oxygenase-1 (HO-1) expression in the interference of fibrogenesis of chronic liver diseases. Over-expression of HO-1 in the liver by rAAV/HO-1 markedly reduced the severity of fibrosis and portal hypertension in a micronodular liver cirrhosis model. This finding was accompanied with improved liver biochemistry and fewer infiltrating macrophages and activated hepatic stellate cells (HSCs) in rAAV/HO-1-transduced livers. The anti-fibrogenic effects of the rAAV/HO-1 approach were partly through the over-expression of HO-1 in activated HSCs. HO-1 and its enzymatic product, carbon monoxide, could suppress the collagen synthesis and the proliferation of activated HSCs isolated from injury livers in a cell culture system. Administration of rAAV/HO-1 to cirrhotic livers could promote the resolution process of fibrotic tissue and attenuate the severity of fibrosis in the presence of etiologic agent for long term. These data provide the direct evidence of enhanced liver HO enzymatic activity in the control of the development of liver cirrhosis, and support the clinical application of rAAV in the treatment of liver cirrhosis.

During the past 30 years make a lot of headways for the basic research of cancer as well as in clinical research . The main headway was displaied at improvement of diagnosis and treatment . In primary liver cancer ( PLC ) particularly early diagnosis and combinative treatment had gained remarkable success . Therefor the prognosis of PLC was remarkable improved also. In 1972 , we had reported clinical analysis of 3254 cases with PLC . These cases all was in hospital patients from 11 provinces of eastern , western , southern and northern in China during 1966 China during -1968 . This data was repeat quoted during the past 30 years . For study the changes of the clinical aspects of PLC we had collected 3250 cases with PLC . They were continuously in hospital patients during 1996-1998 from 10 provinces same 30 years ago. The operative patents and inoperative patents all were included . Their diagnosis was determined by pathological examination (58.8%) AFP , B-US , CT and/or MR . The clinical data was analyzed and compared with the data of 30 years ago .

The 3250 patients were aged 1-80, with an average age of 49.1 years, older than 30 years ago (43.7 years) , (p<0.05) .The male to female ratio was 2.3:1 , lower than that 30 years ago (7.7:1) , (p<0.01) . The HBsAg positive rate was 81.0 % , the HCV-Ag positive rate was 13.2% . The AFP positive rate was 75% (>20 ng/ml) and 44.6% (>400ng/ml) . However the AFP positive rate (>400ng/ml) was 65.7% at 30 years ago (p<0.05) . The early , median , and late stages accounted for 29.9% , 51.5% and 18.6% respectively in comparison with the rates of 0.4% , 47.0% and 52.6% 30 years ago (early and late stages p<0.01, median p> 0.05% ) . The overall resection rate was 46.3% (early stage 78.3% , median stage 44.5% , late stage 0% ) more then that 30 years ago (6.1%) , ( p < 0.01) . The 1, 3, 5 years survival rate were 66.1% , 39.7% and 32.5% respectively for the whole group (93.5% , 70.1% and 59.1% respectively for the early stage patients) .However, 30 years ago half year survival rate and 1 year survival rate only were 36.1% and 10.5% respectively for the whole group ( 1year survival P < 0.01) .

In comparison with the situation 30 years ago , a lot of patients can be diagnosed earlier . More PLC undergo resection. The prognosis of PLC has been improved greatly. Of course, these cases all are in patients that can not explain overall picture of Chinese cases with PLC . However comparison with 30 years ago the clinical aspect of PLC have may improved that least in same to in hospital patients are confirmation. Conventionally, predictive factors for metastasis and relapse after the surgical treatment of hepatocellular carcinoma (HCC) consist morphological (both gross and histological) features of the tumor, including TNM staging, tumor cell grading, capsule, and vessel invasion, etc. However, sometimes, these can not exactly reflect the biological features of HCC and patients' outcome. A complementary way is to analyze molecular markers (biomarkers) for their significance with reference to metastasis and recurrence of cancer. In recent years, with the development of cellular and molecular biological techniques, many biomarkers related to invasion, metastasis, recurrence and survival have been explored in authors' institute. Many biomarkers have been found to be related to the invasiveness and metastasis of HCC. One group is positive related factors, including p16 and p53 mutations, H-ras, c-erbB2, mdm2, TGF-a, epidermal growth factor receptor (EGFR), MMP-2, uPA, uPA-R and PAI-1, ICAM-1, VEGF, PD-ECGF, bFGF, and osteopontin (OPN), etc. The other group is negative related factors, including nm23-H1, Kai-1, TIMP-2, integrin a5, E-cadherin, etc. These factors could be potential predictive markers for the metastasis and recurrence of HCC. Serum VEGF, ICAM-1 and PAI-1 levels were higher in patients with metastasis than those without metastasis, while serum Thrombomodulin concentration negatively associated with the intrahepatic spreading and portal vein thrombi of HCC. Overexpression of MMP-2, VEGF, TGF-a, and EGFR in HCC tissues, and LOH on chromosome 14q (D14S62 and D14S51) in plasma DNA were also related to metastatic recurrence of HCC patients. p53 mutation or nuclear accumulation of p53 expression could be a valuable marker for predicting the prognosis of HCC patients after resection. E-cadherin, nm23, TIMP-2 are promising prognostic markers.

In one previous study, using comparative genomic hybridization (CGH), we found loss on chromosome 8p was more frequent in metastatic lesions than the matched primary tumors of HCC, which indicated 8p deletion might contribute to HCC metastasis. To narrow the location of metastasis-related alteration regions, we analyzed 22 primary and matched metastatic lesions of HCC by genome-wide microsatellite analysis. Many regions with increased-grade AI in metastatic lesions were 4q21-22, 4q32-qter, 8p23.3, 8p11.2, 8q24.1, 9p11, 9q31, 11q23.1, 13q14.1-31, 13q32-qter, 16p13.3, 16q13, 16q22, 17p11.2-13.1 and 19p13.1, which were considered metastasis phenotype related. Among of them, loss on 8p was again proved related to progression and metastasis of HCC, and 8p23.3, 8p11.2 were two likely regions harboring metastasis-related genes. These provide some candidate regions for further study to identify putative metastasis suppressor genes of HCC. Recently, we constructed a specialized small chips with 100 cDNA clones of chromosome 8p (all 83 ESTs on 8p from the unified database and 18 genes from HCC cDNA library), and compared their expression difference between the cell clones with different metastatic potentials (MHCC97-H and MHCC97-L). Eleven of them were found to be significantly down-regulated in the MHCC97-H cells with higher metastatic potential. One full length gene, HTPAP, was obtained from one differently expressed EST. In vitro study, we found the HTPAP gene transfection could significantly suppress the invasion activity of HCC cell. The in vivo functional analysis of this gene is performing.

More recently, we collaborated with NCI/NIH (USA) to analyze the expression profiles in HCC from 40 patients without or with accompanying intrahepatic metastases. Based on their gene expression signatures, using a supervised machine learning algorithm approach, we generated a molecular signature that correctly classified patients with or without accompanying intrahepatic metastases and have identified genes that are mostly relevant to the prediction outcome including patient survival. Osteopontin (OPN), a secreted phosphoprotein, was found to be overexpressed in primary HCC with intra-hepatic metastasis and a neutralizing antibody against osteopontin can block invasion of highly metastatic HCC cells in an in vitro assay of invasion, and suppress the lung metastasis of nude mice model (LCI-D20) bearing human HCC. These data identify osteopontin both as a prognostic marker and potential therapeutic target for HCC.

Immuno-gene therapy, which mainly consists of cytokine gene therapy and gene-modified tumor vaccine, is an important component of gene therapy for hepatocellular carcinoma (HCC).

There were two strategies for application of cytokine gene therapy. First, cytokine gene was transduced into immune effector cells (TIL, LAK,.CTL and macrophages) and transferred to target tissues, thus increasing local concentration of cytokine and more effectively enhancing anti-cancer immunological response. Some cytokine genes including IL-2, IL-12, IFN , TNF and GM-CSF were applied in gene therapy for HCC. Secondly, cytokine gene and (or) co-stimulating molecule gene were transfected into cancer cells, resulting in high expression of cytokines and MHC molecules in microenvironment around tumor tissues, and enhancement of immunogenicity of cancer cells, thus effectively activate specific immunological response against cancer. Transfection of cytokine genes and B7-1 gene into HCC cells, to increase immunogenicity, decrease tumorigenecity, and induce host specific anti-cancer cytotoxic T lymphocytes, could effectively prevent invasion from the parent cancer cells and showed some therapeutic response in nude mice model bearing human HCC. Cancer vaccine with combined transfection of IL-2 and B7-1 genes induced more strong immunogenicity in HCC cells, effectively induced systemic anti-cancer immunity and prevented the attack of implanted mild parent cancer cells, indicating that cancer vaccine with combined gene transfection may become a therapeutic approach to prevention of metastasis and recurrence of HCC.

The current trend of cytokine gene therapy is focused on combined cytokine gene therapy and targeted tissue expression. Combined cytokine gene therapy may induce synergetic biological effect and increase the effectiveness and safety of gene therapy. Combined application of IL-2 and IFNa genes can prevent and inhibit metastasis and recurrence in HCC. A fusion gene with IL-2 and IFNa was established in our institute, expressed fusion protein with the cytokine functions of both IL-2 and IFNa, and enhanced combined anti-cancer effects of both cytokines. Target expression was investigated drawing assistance from antibodies against cancer cells, ligands with specific surface marker of cancer cells, and vectors expressing cancer-targeting genes with specific transcriptional regulatory sequences. Combined transcriptional regulatory sequences with -fetoprotein enhancer and albumin promoter have been designed and cloned in our institute to regulate specific expression of cytokines in HCC cells and to enhance inhibitory effect on HCC cells.

Approaches to strengthening specific host immunity against cancer via dendritic cells (DCs) have become a new strategy in cancer biotherapy, which include: (1) DCs were in vitro pulsed with cancer antigen peptides, tumor lysates, or tumor RNA, and infused with tumor cells for in vivo injection; (2) DCs were transduced with tumor specific, or tumor associated, antigen which was mediated with viral or non-viral vector; (3) immunomodulation of cytokines and chemokines combined with DCs can induce more effective anti-cancer immunity. Transduction of tumor antigen gene into DC resulted in target expression of respective tumor antigen, prolongation of immunological stimulation, and induction of specific response of cytotoxic T lymphocytes, indicating a valuable protocol for preparing gene-engineering cancer vaccine.

Our experiments showed that DC modified with MAGE-1, IL-12 or Flt3 ligand (FL) genes can induce in vitro effective killing for HCC cells, indicating it may become a new vaccine applying in the prevention and treatment of HCC. Treatment with functional gene-modified DC tumor vaccine will be a new approach to immuno-gene therapy for HCC.

The mortality of HCC in China fluctuated between 20-24/100,000 recent year, ranked second of overall cancers. The deaths of HCC in our country accounted for 43.7% in the world. According to the epidemiological studied, HBV, AFB1 and water contamination are three important environmental risk factors. In Qidong-Haimen area people drank water from Yangtze River with HCC mortality 20 per 100,000, people drank pond-ditch water with HCC mortality 100 per 100,000 and people drank deep well water with HCC mortality 10 per 100,000. After Meta-analysis, results from six case-control studies showed that Odds-Ratio for drank pond-ditch water was 2.46 (95% CI 1.69-2.59), Population Attributable-Risk (PAR) was 30.39% and OR for drank deep well water was only 0.75 (95% CI 0.55-1.03). During 1970s, there was a hypothesis of H 2 O+X which water was nonsense, but X substance might associate with liver cancer, especially in the pond-ditch water. After the thousand of ELISA test for contents of Microcystin (MC) in water, there were 294 pg/ml in pond-ditch water, 284 pg/ml in small river water and less then 50 pg/ml in deep well water (minimum level detectable).

According to the age distribution of liver cancer, after 20 year latent period the exposure curve was started in the very beginning of life. Another hypothesis was set down that liver cancer developed in the early year of life. A group of SD Sprague-Dawley rats were injected with MC-LR in their period of embryo to study the effects on placenta and embryo development. The results showed that MC-LR could pass through placental barrier of pregnant SD rats, damage their fetus liver and kidney and developed malformation of fetus rats with dose-response effect. Another experiment for determination the tumourigensis of cyanotoxins (microcystin, MC & Nodularin, Nod). Using more than 100 HBVx transgenic mice and same quantity of controls (all 7 d old) injected to AFB1 6mg/Kg.bw once at first week and followed by MC and Nod at 10 mg/kg.bw once a week from weeks 2-16 by intraperitoneal rout. The mice were sacrificed at the end of 16 weeks and 52 weeks and examined histopathologically. Results were in the following table and graph.

Our results suggested that the predisposition effects of MC and Nod were prominent in the presence of HBV infection and AFB1, where the HCC induction reached 20-35%. Nod might have direct carcinogen and initiation effect. MC and Nod combined together and interacted with HBVx got higher HCC prevalence rate. After one year, second group of transgenic mice were sacrified. The prevalence of HCC in the 52 weeks occurred more than double than in the 16 weeks.

Three main environmental risk factors were interacted each other. However, more importance risk was HBV combined with others, especially combined with Methylentetrahydrofolate Reductase (MTHFR677 with genotype-Any T). The non-combined Odds Ratio (OR) of liver cancer was 17 (95% CI 5.10-59.90) and the combined OR=41 (95% CI 8.20-212.20) (Figure 2) .

The control strategy for liver cancer was control of water, change to rice and prevention of hepatitis (7 Chinese characters). 

Background Early Hepatitis C viral (HCV) kinetics was previously shown to predict sustained virological response (SVR 

For HCV we find that a composite 2 nd phase slope criteria can predict SVR with NPV=100% and specificity=34% already after 4 weeks of treatment. Furthermore, prediction with NPV=100% and specificity=28% can be obtained already at 1 week. Combining both criteria allows to increase specificity up to 50%. Moreover, by selecting more strict thresholds for the above criteria one obtains PPV=94%. For HBV two major patterns were identified during weeks 4-48: a flat second phase (RF) or a slow second phase decline (RS) in 35% and 65% of patients, respectively. This slow second phase was then followed by three different third phases: HBV DNA became undetectable (<400copies/ml) (RSBD) in 37%, or reached a flat third phase (RSF) in 35%, or had a staircase pattern (RSFS) in 28% of RF patients. No patients with RF pattern as identified at 48 weeks lost HBeAg during that same period, compared to 28%, 43% and 77% of the RSF, RSFS and RSBD groups. However, when the RF pattern was identified at 16 weeks we find that 10% of early RF patients had HBeAg loss at 16-48 weeks.

HCV sustained viral response to treatment with peginterferon-alfa and ribavirin can be predicted, independent of genotype, as early as 1 or 4 weeks of treatment. On the other hand, the earliest HBV kinetics can predict HBeAg loss is at 16 weeks. Moreover, changes in HBV kinetic patterns occur even after that time making the prediction of end-point less accurate. This may indicate that HBV has more dynamical viral dynamics as compared to HCV, and that processes triggered later on during HBV treatment can influence response to treatment, while the fate of HCV treatment is determined by early kinetic events.

Chronic infection with hepatitis C virus (HCV) is characterized by a dynamic equilibrium between virus production and clearance. A characteristic early decline of serum HCV RNA is observed when this equilibrium is disturbed by interferon a. Mathematical analyses of the viral decline during the initial phase of interferon treatment resulted in the estimation of a very short half-life of free hepatitis C virions in vivo (< 5 h), and indicated that the rapid first phase (day 1) of the initial decline relates to the decay of free virus particles, whereas the much slower second phase (≈ day 2) of the initial decline reflects clearance of productively infected cells. Both first-phase und second-phase slopes and the extent of viral decline after 24 hours have been shown to depend on HCV genotype and to be larger for patients with HCV genotype non-1 than for patients with genotype HCV-1. A significant improvement in sustained virologic response rates has been achieved in HCV-1 infected patients by the long-acting pegylated interferons. Kinetic analyses indicated that treatment with peginterferon a may reinforce the death rate of infected cells particularly in HCV-1 infected patients and/or stabilize the therapeutic effect on viral production. These kinetic analyses are central to our current understanding of antiviral therapy and have influenced approaches towards treatment of patients with chronic viral hepatitis.

Hepatitis B virus (HBV) infection is a global health problem and the clinical outcome of chronic HBV infection depends on the frequency and severity of hepatitis flares in the immune clearance phase, and several factors including viral, host or environmental ones are reported to affect the clinical outcomes of patients with chronic HBV infection. By phylogenetic analysis of hepatitis B viral (HBV) isolates, 8 different genotypes (A to H) have been recognized worldwide. Most of the HBV genotypes have a specific geographic distribution, with predominance of genotypes A and D in North America, Western Europe and India, B and C in the Southeast Asia and Far East including Taiwan, and F in South America. The distribution of genotypes E, G and H is less well studied. The impact of HBV genotypes on the clinical aspects of HBV infection including clinical outcome of chronic infection and therapeutic response to antiviral treatments has been clarified. In Taiwan, genotype C is associated with more severe liver disease including cirrhosis and hepatocellular carcinoma (HCC), and genotype B is associated with the development of HCC in young non-cirrhotic patients. In contrast, genotype B has a relatively good prognosis in Japan and China and is rarely associated with the development of HCC. Similarly, genotype D is associated with more severe liver disease than genotype A in India and may predict occurrence of HCC in young patients. Serologically, genotype C tends to have a higher frequency of hepatitis B e antigen (HBeAg) positivity and a higher serum HBV DNA level than genotype B. In addition, genotype C patients, compared to genotype B patients, have a delayed HBeAg seroconversion in the immune clearance phase of chronic HBV infection. Although superinfection of HBV on top of hepatitis B carriers occurs, it is rarely associated with acute exacerbations. Virologically, genotype C bears a higher frequency of core promoter mutation than genotype B, and patients with core promoter mutation are more associated with the development of HCC than those without, which apply to both genotypes B and C. As to the response to antiviral treatment, genotypes C and D are associated with a lower response rate to interferon therapy compared with genotypes B and A. Compared to genotype C, genotype B may have a more durable HBeAg response to lamivudine therapy. However, both genotypes have a similar virologic response and risk of developing drug-resistant mutants after 1 year of therapy. These lines of evidence from different Asian countries have lent strong support to possible pathogenic differences between HBV genotypes B and C. In Western countries where genotypes A and D prevail, the clinical and virologic studies also indicated the long-term outcome of chronic hepatitis B is different in patients infected with HBV genotype A, D, or F. These data further help us in understanding the whole picture of patients infected with different HBV genotypes worldwide, and highlight the importance of determining the genotype in patients with chronic HBV infection in our future clinical practice.

Department of Medicine, University of Indonesia, Jakarta, Indonesia A few years recently, Indonesia has a very fast increase in drug user, particularly the use of heroin intravenously (IDU). This can be viewed as a major source of hepatitis C virus (HCV) transmission which can change previously reported route of HCV transmission inIndonesia.We study 200 patients hospitalized for rehabilitation program, 167 (83.1%) were IDU. Collected sera were kept at minus 40 C until use. Most of the patients were male (90.6%) and the mean age of them compared to 23.1 % in non-IDU. Anti-HCV were positive in 136 IDU patients (81.4%) and HBsAG positive in 27 IDU patients (16.1%). HCV genotype ( in the first 100 consecutive patients) were 1a 57.5%, 1b 10%, 2a 10%, 2b 15%, and 3a 7.5%. Risk factors for HCV transmission for these patients were exchange needle and also tattoo. Fortunately in IDU patients, therapy with interferon in combination with ribavirin gave a better result ( 100 % EOT) compared to non-iDU chronic hepatitis C patients (72.2%). In summary, transmission of HCV and HBV in IDU patients was high, however, the response to therapy was better.

There is no consensus on the management of lamivudine resistant HBV. Long term treatment studies have reported the occurrence of HBe seroconversions in patients with lamivudine resistant HBV. Most HBe seroconversions however occur late preceded by multiple episodes of hepatitis flares and accompanying disease progression. Most physicians will not stop therapy if ALT is not persistently raised. This usually occurs when the mutated species has not been completely selected. When ALT becomes persistently raised, the opinion on further management is divided. Switching to or adding other therapies including interferon, thymosin and adefovir, either as monotherapy or in combination with lamivudine have been shown to be beneficial.

Prevention of the occurrence or delaying occurrence of these drug resistant HBV strains can be effected by being more stringent on the initial indication for lamivudine therapy. In young patients with no evidence of liver cirrhosis or decompensation, it could be given selectively to those who have a higher chance of seroconversion with short duration of treatment i.e. those patients with significantly raised ALT levels. Should HBe seroconversion failed to occur in these patients lamivudine can be discontinued and treatment reinstituted when required. An exception to this rule would be if there is continuing liver inflammation in an individual with decompensated cirrhosis.

Combination therapy with pegylated interferon and lamivudine appears to give a higher rate of HBe sreroconversion than lamivudine monotherapy and less incidence of lamivudine resistance. Combination therapy using lamivudine and adefovir has not been shown to be superior to monotherapy using either agent although this combination is likely to be the most successful way to control drug resistance.

A number of other drugs are being evaluated and these including Entecavir, Cleuvudine and Telbivudine would add to the armamentarium of useful agents in the battle against drug resistant HBV species.

Large, multicentre, Phase II and III studies of nucleoside analogues against HBV (Lamivudine, Adefovir, Entecavir and Telbivudine) have demonstrated that serologic response in HbeAg+ chronic hepatitis B (CHB) increases with duration of therapy. However, prolonged therapy may be associated with increased viral breakthrough: 15% after 1 year lamivudine; 65% after 1 years lamivudine. The decision to continue long-term therapy is dependent on multiple factors: (1) efficacy (both during and post treatment) including virologic, serologic and histologic responses; (2) safety, including risk of cumulative toxicity, and risk of flares during treatment (from viral breakthrough) and posttreatment (from viral rebound). Other important factors, which may vary between countries, include patient compliance, cost (depending on reimbursement) and availability of longterm monitoring.

Attempts to construct an optimal profile for therapy have relied on identification from Phase III studies of reliable predictors of both response to therapy and also of risk of breakthrough. However, the cost-effectiveness of such limitations on treatment is unproven. However, recent data would suggest that life-long therapy is indicated in all DNA+ cirrhotic patients regardless of serum ALT levels.

Finally, recent studies have suggested the duration of therapy continued after successful HbeAg seroconversion may determine the durability of response post-treatment. Minimum duration should be 6 months with best results obtained after 18 months.

In future, the optimal duration of therapy will depend very much of efficacy and safety of new antiviral therapies. There is little doubt that future combination therapies will significantly reduce the risk of breakthrough, albeit at an increased cost. Continuing therapy until at least 6 months post-HbeAg seroconversion in HbeAg+ CHB and life-long in all HbeAg neg CHB may become the gold standard in those who can afford this.

It is estimated that 400 million people are chronically infected with the hepatitis B virus (HBV), 75% being Asians. Current treatment for patients with HBeAg-positive CHB suppress HBV replication, reducing HBV DNA levels to undetectable levels by PCR in 20-30% and resulting in HBeAg seroconversion in 12-20%. Suppression of HBV replication results in improvement in liver biochemical and histological parameters including retardation of, and in some cases, even reversal of fibrosis. Treatment needs to be more precise and tailored to suit patient types. Information on the natural history, genotype, HBeAg status of patients, of co-infection with other viruses, underling medical condition and others can help determine response rates. HBV Genotypes may help determine choice of therapeutic agents as some types appear to respond better with immunomodulatory agents. It is becoming clearer now that the most important factor that cause disease progression is prolonged, low level viremia. The challenge is to choose the most appropriate patients and provide them with the most appropriate treatment, given the limitations of the current therapeutic modalities and evolution of new drugs and information on factors that may determine treatment response. Treatment of HBeAg-positive CHB is either with Lamivudine (LAM) or Interferon. LAM is the best known NA but has limitations especially the development of YMDD resistant mutants. Other drugs such as Adefovir and Tenofovir have emerged to treat LAM-resistant mutants. Treatment of HBeAg-negative Chronic hepatitis B patients is also challenging. Earlier treatments suppress HBV replication and biochemical or histological activity but seldom attain sustained responses. Long term treatment may result in a more sustained response in some patients (< 20%). At present nucleoside analogues (NA) that do not cause resistance in the long term may be an option for this group of patients. NA can be considered for patients with decompensated cirrhosis. NA can prevent decompensation in patients with compensated cirrhosis. Trials of Pegylated IFN are in progress, both as monotherapy as well as combination. Results of these ongoing trials will have an impact on the future role of IFN in CHB. Other agents that are being studied include Entecavir, Emtricitabine, Telbivudine, and Valtocitabine. Combination treatment will be expected to be beneficial as a means of limiting resistance and achieving additive or synergistic efficacy. Long term therapy will be useful with agents that are potent, safe, tolerable and low resistance.

Treatment for chronic hepatitis C (CHC) is becoming more precise with the availability of information of the HCV genotype and the viral load. Tremendous progress has been achieved with the treatment of CHC where the likelihood of "cure" is greater than the chance of failure. Combination treatment using Pegylated IFN and Ribavirin for 24-48 weeks is now the standard treatment for patients with CHC. While sustained viral response rates (SVR) have always been the used to decide on the success of treatment, early viral response rates (EVR)i.e. assessment at 12 week of treatment can guide management decisions especially with in patients with genotype 1.

Hepatitis B virus (HBV) is a serious infectious and widespread human pathogen (1). Chronic HBV infection has a high risk to evolve into hepatocellular carcinoma. At present, the detailed pathogenesis of HBV infection is still unclear and a definite diagnosis of HBV liver necroinflammation still relies on biopsy histological examination (2, 3). We use proteomic technology to systematically analyze HBV-infected serum samples aiming at searching for disease-associated proteins that can be used as biomarkers with clinical value and as molecular targets for pathogenetic study.

We studied 20 chronic hepatitis Be antigen positive Chinese subjects from Queen Mary Hospital, Hong Kong. Ten were in the immune-tolerant phase (Group 1) and ten were in the immune-clearance phase (Group 2). Group 1 patients had low ALT levels and low necroinflammatory scores (LNS) and group 2 patients had high ALT levels and high necroinflammatory scores (HNS). We used both 2D-gel / MELDI-Tof-MS and ProteinChip / SELDI-Tof-MS proteomic technologies to analyze the serum samples. The resulting data were compared to those from normal serum samples, and were statistically evaluated.

Compared to control normal samples, several proteins or polypeptides were found differently expressed in HBV-infected sera, as shown by 2D gel protein profiling. These proteins have molecular weights ranging from 15 -55 kDa. Complementarily, another several protein or polypeptide peaks with molecular weights from 4 -28 kDa were detected to be significantly different in HBV inflammation by means of ProteinChip / SELDI-Tof. Some of these protein alterations can be used to differentiate HBV infection from non-infection, and the others can be used to discriminate low score from high score necroinflammation.

The availability of the human genome sequence has enabled the exploration and exploitation of the human proteome. Because proteins, once synthesized on the ribosomes, are subject to a multitude of post-translational modification steps, the number of different protein molecules expressed by the human genome is probably closer to a million in comparison with the hundred thousand genes generally considered by genome scientists. Due to the complexity, diversity and polymorphisms of proteins, the genomic information does not allow the efficient prediction of all the activities and functions of proteins. The more and more proteome projects were therefore initiated worldwide.

The liver is the largest organ in the body, probably second only to the brain in organ complexity. It displays the important functions for the digestion, metabolism, production of red blood cells during embryonic development, production of various plasma proteins, detoxification of xenobiotics, phagocytosis of solid material, and synthesis of fatty acids. Liver has central roles in activation, catabolism and excretion of retina, and in growth, reproduction, cell proliferation, differentiation and integrity of the immune system. Liver diseases, such as viral hepatitis, liver cancer, alcoholic liver injury, and drug-related liver injury, are critical challenges for human beings and great challenges for modern medicine. There was broad interest and strong voice for a Human Liver Proteome Project (HLPP). HLPP aims to identify all the proteins in human liver as well as the amount, location, interactions, and activity of each protein. The overall goal of HLPP is a comprehensive analysis of proteins in human liver under health and disease conditions. The establishment of the knowledge of complete biological functions of liver will provide the tools to allow the development of new therapeutic approaches for human liver diseases.

The scientific activities of HLPP include 1. Collection and banking of human liver tissue specimen. 2. Generation of compiled expression profile of human livers in health and disease states. 3. Elucidation of protein modification profile. 4. Establishment of subcellular localization profile. 5. Networking of protein interaction linkage maps. 6. Development of antibody bank for human liver proteins; 7. Bioinformatics of human liver proteome. 8. Integration and correlation of human liver proteome with liver transcriptome and human genome. 9. Comparison and elucidation of proteome alterations of human livers under different disease states.

The Resource development goals include 1.entification of sources of liver samples for analysis to be shared among investigator groups for comparative analysis of standard specimens.2. Provision of one or more reference specimens. 3. Development and assessment of tools for liver protein analysis, including creation of antibody panel for liver proteins and further antibody protein arrays.4. Development of a format for database(s) of liver protein expression, liver protein interaction map, protein modification profile, subcellular proteome, bridging of liver transcriptome and its proteome with human genome, and integration of liver

The term "proteomics" was first coined in 1995, to describe the large-scale characterization of the entire protein components of cell type, tissue or whole organism. Proteomics studies the global protein expression profile instead of the behavior of a single protein. Systematic analysis of the function of genes can be performed at the nucleic acid (gene transcript) or protein level. The latter has the advantage of being closest to function, since it is proteins that carry out most of the activities in the cell. For proteomic analysis, cellular proteins can be resolved by 2-D electrophoresis , and then subjected to mass spectrometric analysis using matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. Identification of specific proteins can be performed using the SWISS-PROT protein database. Mass spectrometry can now identify proteins with a very high sensitivity and high throughput. New approaches include the isotopic labeling of proteins to obtain accurate quantitative data by mass spectrometry, methods to analyze peptides derived from crude protein mixtures, and approaches to analyze large numbers of intact proteins by mass spectrometry directly.

The single most common application of proteomics is protein identification (protein expression profiling). However, the aim of proteomics is not only to identify all the proteins in the cell, but also to create a complete three-dimensional map of the cell indicating where the proteins are located (structural proteomics) and what functions they perform (functional proteomics). In addition, proteomics can also be used to study protein-protein interaction, post-translational modifications, and proteome mining. The proteomic data will help to elucidate the functions of proteins in the pathogenesis of diseases and the ageing process, and could lead to the discovery of novel drug target proteins and biomarkers of diseases. Recent advances in proteomic technology and the potential applications of proteomics in biomedical research will be further discussed.

Emerging from the genomic era in the 21st century are functional genomics, proteomics and bioinformatics. The Centre for the Study of Liver Disease (CSLD) in collaboration with several other institutions have embarked several major programs to develop and sustain progress in proteomic research in clinical medicine with special interest in hepatitis and liver disease. Hepatitis B virus (HBV) and its related complications in liver diseases (cirrhosis and HCC) remain a global health problem and one of the leading causes of morbidity and mortality in Hong Kong and around the region. Liver cancer remains the second commonest fatal cancer in Hong Kong, while hepatitis B viral infection afflicts 10% of the population. With the advents of human genome sequence data, proteomic technology has revolutionized and accelerated the identification of disease-related biomarkers and delineation of the key molecular and cellular pathways involved in liver diseases and cancer. Information on human tissue proteome, pharmaproteome and biomarker discovery are essential for further advancement of prevention, diagnosis, prognosis and treatment for patients with non-curable human diseases. The presentation will give an overview of local achievements using proteomic approaches in liver disease that are difficult to attain in the past. (Supported by CSLD and Research Grants Council of Hong Kong)

Chronic Hepatitis C (CHC)

Liver Biopsy has been recommended in management of CHB and CHC by various consensus conferences, as biopsy provides prognostic information about the natural history and predicts the likelihood of response to antiviral therapy. 1-6 Only 1/3 rd of patients chronically infected with HBV or HCV develop serious complication like liver cirrhosis, liver cell failure and hepatocellular carcinoma leading significant morbidity and mortality. 1-6 Though many studies have demonstrated that risk factors associated with cirrhosis progression in patients with chronic viral hepatitis are age, regular alcohol consumption, co-infection with other hepatitis viruses. HIV coinfection; necroinflammatory changes 7-12 and fibrosis on histological examination is reliable predictors of progression and response to therapy irrespective of host factors and viral factors like viral load and genotype. 13-14. Hence liver biopsy has remained "Gold Standard" in the management of chronic viral hepatitis. In addition to necroinflammatory reaction and fibrosis liver biopsy can give additional information like steatosis, iron content, histological response to antiviral therapy, which could affect the management of patients with chronic viral hepatitis. 15-16 . Patients with severe histopathological changes might benefit from prolonged treatment with antiviral if they have not responded to initial treatment 17 . Identifying factors associated with histological response to treatment may be useful in guiding ongoing antiviral therapy.

Limitations of liver biopsy are (1) invasive technique (2) cost of the biopsy (3) sampling error (4) intra and interobserver variability. Liver biopsy can be associated with potential complications like pain in almost 30% patients while severe complication like bleeding in 0.3% and death in 0.03% patients 18 . Around 10 to 20% sampling error occurs in biopsy and there is 15 to 30% chance of underestimating cirrhosis in percutaneous biopsies. 19 Intra and interobserver agreement is seen in 60 to 90% of cases. 20 Liver biopsy estimates the static mass of fibrosis and may not assess dynamic process of constant degradation, formation and remodeling. Because of above mentioned problems there is reluctance in accepting liver biopsy by patients and many of the physicians.

Many studies have evaluated surrogate markers for histology in distinguishing cirrhosis from no cirrhosis, because moderate to severe fibrosis is often histological variable on which treatment decisions are based. 19 . Simple, inexpensive and reliable noninvasive means to assess disease severity in chronic viral hepatitis are being explored. Among the routine laboratory parameters decreased platelet count, increase AST/ALT ratios, prolonged prothrombin time are the earliest indicators of cirrhosis. Serum markers of fibrosis like products of collagen synthesis/degradation PIINP, Type IV collagen, extra cellular matrix enzymes like TIMRI MMP, hyluronic acid and Laminin have limited accuracy in predicting fibrosis. 19 Combination of biochemical markers, alpha 2 macroglobulin, apolipoprotein A1, hepatoglobulin, GGTP and total Bilirubin (fibrotest) and ALT (actitest) have been shown to be accurate noninvasive markers in chronic viral hepatitis 21,22 If these findings are confirmed by large trials in various parts of the world, need for liver biopsy will go down.

When we evaluated 105 patients with chronic hepatitis B severity of Liver disease did not correlate with age, "e" antigen status and transaminases values 23 . Similarly while evaluation natural history of chronic hepatitis C infection. We could not find very good correlation between genotype, viral load and transaminases. 24, 25 As performing liver biopsy is not possible on each and every patient of chronic viral hepatitis doing liver biopsy in select group of patients is practicable. Those patients with evidence of portal hypertension on imaging and endoscopy may not need biopsy. Patients with persistently normal liver enzymes and normal imaging who are asymptomatic are unlikely to be benefited by treatment and very less likely to have severe disease may not need biopsy. Patients who are unwilling for therapy need not be subjected to biopsy. Patients who meet biochemical and virological criteria for treatment but unlikely to undergo liver biopsy should be offered treatment without biopsy. Patients who are eligible for treatment and willing for biopsy should be subjected for liver biopsy. If patients is uncertain regarding therapy and only wishes to take treatment if disease is severe should be subjected for biopsy.

We analyzed 98 liver biopsies of patients with CHB and 48 patients with CHC. Out of 98 patients 69 had mild fibrosis, (metavir score 0-2) and 29 severe fibrosis (metavir score 3-4). Those with mild fibrosis showed mild necroinflammatory reaction while those with severe fibrosis had severe necroinflammatory reactions. On univariate analysis of the variables, age, serum Bilirubin, platelet count, GGTP, SGOT, SGPT, SGOT: SGPT ratio more than 1, HBeAg positive and HBeAg negative, only following variables -GGTP, platelet count and SGOT: SGPT ratio were significantly different in patients with severe fibrosis when compared with mild fibrosis while age, platelet count, SGOT were significantly different in patients with severe necroinflammatory reaction when compared with patients with mild necroinflammatory reaction. On multivariate logistic regression analysis GGTP and platelet counts were predictors of severe fibrosis with overall predictability 84.7%. Out of 48 liver biopsies of the patients with CHC 27 had mild fibrosis and 21 had severe fibrosis, 32 had mild necroinflammatory reaction and 16 had severe necroinflammatory reaction. On univariate analysis SGOT to SGPT ratio and platelet count were significantly different in patients with severe fibrosis as compared to mild fibrosis. On multivariate logistic regression analysis platelet count had overall predictability for severe fibrosis of 75%.

Along with the accomplishment of human genome sequencing, the functions of at least 40%-60% of the genes still need to be characterized in proteome research. Proteome research, which is also called functional genome, can be defined as studies on proteins in whole-cell scale. It has now become a new frontier in biological research. The overall scientific goals of proteome research mainly involve three aspects as follows: 1

973" were carried out in succession undertaken mainly by military academy of medical science, Chinese academy of science and Fudan university. Till now many proteome research centers or laboratories including our lab bloomed in china China is a main epidemic area of liver diseases. Based on the statistics from WHO, there are more than 350 million hepatitis B virus-carriers in the world, and 150 million of which are in China. Besides, 30 million Chinese are hypertitis C virus-carriers. Research has showed that liver cancer is closely associated with such virus infection. Liver cancer is one of the most malignant diseases which result in high mortality. There are nearly 1 million new cases of liver cancer per year in the world and half of which is in China. Approximately 450,000 patients died of liver cancer in our country yearly. New development in proteomics will shed light on the pathogenesis of liver cancer and provide new targets for its therapy. Our current studies of liver cancer proteomics focus on: 1

Reveal the transformational mechanism of liver cancer related proteins, LPTS and LPTSL Lisl and correlative proteomics study

Establish transgenic mouse hepatocellular carcinoma model by targeted intergration of HBsAg and HBx genes into the genome and carry out comparative proteomic study of the liver tissue and serum to identify the key molecules and mechanism related to this HBV gene induced HCC genesis in the model

Liver biopsy is recommended prior to the definite treatment in majority of the patients with chronic hepatitis B and C according to many worldwide guideline. Even liver biopsy is a safe procedure but remain potential risk of minor complicate (pain), major complication and death in about 30%, 0.3% and 0.03% and also high cost. Moreover sampling error can occur in 10-20% and inadequate size of specimen strongly reduces the grading and staging of chronic viral hepatitis. According to these limitation factors, the accurate non-invasive markers to determine the grading and fibrosis are promising.Normally, physical examination, routine blood test (CBC, PT, LFT), and radiological images (Ultrasound, CT-Scan) can easily detect terminal stage of disease. The serum ALT level over 2.5 times ULN have 54-80% sensitivity and 65-87% specificity for the detection of histologic score greater than A1F1. The AST:ALT ratio > 1 is relatively insensitive and may not be a diagnostic for the presence of cirrhosis in patients with chronic HCV infection but highly specific. With the combination of platelet count < 150,000/mm 3 and AST:ALT > 1 can identify patients with severe fibrosis or cirrhosis with positive prediction value 93%, sensitivity 41%, specificity 99%, and negative predictive value 85%. The scoring system combining age, GGT, cholesterol and platelet count proved useful to identify patient without significant fibrosis.The new combination parameter of a 2 macroglobulin, a 2 globulin (or haptoglobin), g globulin, apolipoprotein A 1 , g glutamyltranspeptidase, and total bilirubin (Fibrotest) and serum ALT (Actitest) has been validate in patients with chronic hepatitis B and C which demonstrated high predictive values for significant lesion. The ideal noninvasive diagnostic test for hepatic fibrosis should be readily available, simple inexpensive, accurate prediction, reproducible, sensitive to the effect of treatment and useful in tracking disease progression.

Available therapies for chronic hepatitis B (CHB) in Asia include interferon alpha (IFN), thymosin alpha, lamivudine (LAM) and very recently, adefovir dipivoxil (ADV).The orally administered nucleoside/nucleotide analogue compounds (LAM, ADV) are potent inhibitors of HBV replication and hepatic necroinflammation. Long-term LAM treatment results in an initial response in 70-90%, however, virologic breakthroughs, i.e., >70% in 5 years, set back initial gains achieved in viral suppression, histologic improvement and may even increase the risks of flares and hepatic decompensation.The introduction of ADV has pushed the management of CHB to a new era as results of recent Phase III trials become available. In 48 weeks of treatment of HBeAg (+) patients, Adefovir 10 g OD achieved 12 % anti-HBe seroconversion rates,. Most importantly, no mutations have been noted in over 460 patients treated. After 96 weeks of therapy, virologic breakthroughs were reported in <2% of patients In HBeAg negative patients, 51% have undetectable HBV-DNA at week 48 of treatment. . In LAM-resistant HBV, ADV achieves a reduction of HBV DNA by 4.04 log(10) copies/mL from baseline. Adding ADV to current LAM treatment once YMDD variants are detected, 52 weeks of combination therapy achieved viral suppression in 85%, ALT normalisation is observed in 31-53%. Similar results were also achieved in ADV treatment of LAM-resistant HBV in pre-and post-liver transplant patients. At 10mg OD, renal toxicity has not been the reason for discontinuing ADV therapy in the trials reported so far. In e-negative CHB, the efficacy of LAM and ADV are almost similar. Currently, the risk benefit profile of ADV is similar with LAM as an initial therapy to CHB. In addition, ADV is a safe and effective rescue drug in the treatment of LAMassociated YMMD variants in compensated, as well as, in pre-and post-transplant patients.

Adjunct Associate Professor, the Chinese University of Hong Kong, Hong KongThe main therapeutic goal for chronic hepatitis B (CHB) is eradication of HBV virus. This will bring about resolution of necroinflammation, cessation of fibrotic process, reduced risk of complication of cirrhosis. Treatment options consist of interferon alpha-2b, lamivudine, and adefovir dipivoxil. There are pros and cons in each of these therapies. Many guidelines on CHB management and treatment have been published. Constant updating is required to address important issues on therapeutic options and indications at different stage of the disease, management plan for drug resistant mutations such as lamivudine resistant YMDD mutants, strategy to enhance durability of response, and therapy in specific subgroups. Monotherapy generally has not produced adequate and consistent viral suppression that impact long term clinical outcome. One of the reasons being the persistence of cccDNA in the hepatocytes despite "response" as defined by serum HBV DNA levels. An effective immune clearance of infected hepatocytes is essential for durable therapeutic response. This may not be readily achieved without immune modulators. Pegylated interferons (Peg-IFN) have been demonstrated to have superior pharmacokinetic profile in sustaining therapeutic level throughout the duration of treatment. The response rate to Peg-IFN monotherapy is higher when compared to the standard interferon. It is easier to administer and no added side-effects have been reported. Peg-IFN used as a monotherapy or in combination with a nucleoside analogue is being intensely studied in different international clinical trials for both HBeAg positive and HBeAg negative CHB. The preliminary results look promising with better sustained response.

2 ND CHINA -JAPAN SYMPOSIUM ON LIVER CANCER

Primary hepatocellular carcinoma is a common malignancy with a high mortality rate worldwide. The major risk factors include alcohol consumption, exposure to chemicals such as vinyl chloride and dietary aflatoxin B1(AFB1), and chronic infection with hepatitis B virus or hepatitis C virus (HCV). The mechanisms by which HCV infection promotes the development of liver disease remain largely unknown.HCV is a positive-strand RNA virus that is remarkably efficient at establishing chronic infection. Chronic infection of HCV for long lasting periods with liver inflammation is likely important mechanism for the development of hepatocellular carcinoma. And, thus, the development of anti-HCV agents is very important to prevent the cancer development.The development of anti-HCV agents has been accelerated by the establishment of cell lines in which HCV genome RNA selfreplicates efficiently (referred to as HCV replicon cells). We have attempted to obtain a candidate for anti-HCV function by screening various drugs that have been currently prescribed to patients with various diseases. Among nearly 100 compounds examined, we observed a suppressive effect of cyclosporin A (CsA), an immunosuppresant, on HCV genome replication. Treatment with 1 micro gram /ml CsA or 100 U/ml IFN alpha (used as a positive control) for 7 days decreased the amount of HCV proteins as well as the amount of HCV RNA to levels undetectable by immunoblot and northern analysis. A similar reduction of HCV protein and RNA synthesis was not observed after treatment with FK506 (1 micro gram /ml) which is another immunosuppresant. These results suggested that CsA inhibited the replication of HCV genome in HCV replicon cells.To demonstrate that the anti-HCV effect of CsA was not specific for the artificially constructed HCV replicon cells but could also be extended to a physiologic HCV replication system, we examined the effect of CsA in a hepatocyte-derived cell line PH5CH8 that was previously shown to be susceptible to HCV infection. PH5CH8 cells were treated with HCV-positive plasma, and the HCV RNA genome titer was quantified by RT-PCR analysis at various times after inoculation. By day 5 postinoculation, the HCV RNA genome titer was approximately 10-fold higher than that on day 1 in the normal condition. However, a significant increase of the HCV RNA genome titer at these time points was not observed in cells treated continuously with CsA or IFNalpha. This result suggested that CsA not only inhibited the replication of HCV genome in HCV replicon cells but also inhibited replication of HCV in cultured hepatocytes.In this symposium I would like to discuss the likely mechanism of CsA to suppress HCV genome replication. To examine the viral factors on the effect of combination therapy with interferon (IFN) and ribavirin on chronic hepatitis C, mutational changes of hepatitis C virus (HCV) RNA before and during the treatment were examined.

The whole HCV genome sequence was determined by cDNA amplification and direct sequencing at 6 months before the therapy, just before the therapy and during or 1 month after the therapy in 16 patients with chronic hepatitis C with genotype 1b and HCV RNA more than 100 KIU/ml.

The mean mutational changes were 15.7± 6.3x10 -3 nucleotide (nt)/site/year and were 13.1± 5.2x10 -3 amino acid (aa)/ site/year during the natural course, and 22.0± 11.6x10 -3 nt/site/year and 16.7± 9.5x10 -3 aa/site/year during the treatment, respectively. HCV RNA sequences from 6 null responders showed lower mutational rates (9.8± 4.3x10 -3 nt/site/year) during the natural course compared to those from 7 relapsers (16.0± 4.5x10 -3 nt/site/year) and 6 sustained virological responders (18.4± 7.5x10 -3 nt/site/year). This tendency was also observed for deduced amino acid changes (P<0.05). The amino acid changes were observed mainly in E2 and NS5A during the natural course of HCV infection. The relapsers showed the additional changes in NS5B after the treatment.

Our study revealed that HCV from null responders might have the potential to be resistant to the mutational drift during IFN and ribavirin combination therapy as well as during the natural course.

The hepatitis B virus X protein (X) has been implicated in the carcinogenicity of this virus as a causative factor by means of its multiple functions in the development of hepatocellular carcinoma (HCC). Based on the previous report that X is located in the mitochondria and induces cell death by transfecting the EGFP-fusion constructs of X, we mapped the aa 68 to 117 of X as a region required for mitochondrial localization, but independent of transactivation function. In the cytochemical analysis the mitochondrial membrane potential was decreased by X association with mitochondria. Furthermore, PTP inhibitors, reactive oxygen species (ROS) scavengers and Bcl-xL prevented the mitochondrial apoptotic pathway operated by X.In this study, we employed the protein transduction method to introduce the recombinant X (rX) or its mutant directly into HuH7 cells in concentration dependent manner. We observed the localization of rX in the mitochondria and activation of Erk activity as well. The level of cellular ATP was decreased after rX transduction. The generation of ROS was observed after incubation with rX. Comet assay showed that incubation with rX caused DNA damage, which was protected in the presence of ROS scavengers. DNA damage was then analyzed by the Big Blue Rat cell line technique. So far, nucleotide transition becomes dominant as compared to the control without rX transduction. Thus, ROS generation by X association with mitochondria plays an important role in the cell transformation by HBV, representing paradigm shift of viral hepatocacinogenesis.

Hepatitis B X-interacting protein (HBXIP) was originally isolated as a human protein which binds hepatitis B X (HBX) protein.HBXIP is widely expressed in human tissues, however its fucntion has been unknown. We show that HBXIP functions as a cofactor of Survivin, an anti-apoptotic protein that is overexpressed in most human cancers. Survivin-HBXIP complexes associate with pro-caspase 9 and prevent its recruitment to Apaf1, and thereby selectively suppressing apoptosis initiated via the mitochondria pathway. Notably viral HBX protein interacts with HBXIP/Survivin complexes and increased their pro-caspase 9 binding. The findings thus provide novel insights into the anti-apoptotis mechanism of Survivin, and suggest a link between the cellular apoptosis machinery and a viral pathogen involved in hepatocellular carcinogenesis.