Immunization of a recombinant plasmid encoding hepatitis C virus core protein

Journal of Gastroenterology and Hepatology (2006) 21 (Suppl. 2) A97

Poster Session—Hepatitis (Diagnosis/Pathogenesis/Therapy)

Conclusion These findings are in contrast with other studied population (Black, Hispanic and Asian like Chinese and Korean)therefore distribution of these polymorphisms are strongly ethnicdependent. Determination of these genotypes is probably not a suit-able genetic marker for the risk assessment of HBV in our subject.

#073

Inhibition of HBV replication by interferon-a viaAAV-mediated gene transferMING-LIANG HE,1 ZHI LI,2 BO-JIANG ZHENG,2

KWOK YUN YUEN,2 MARIE C LIN,3

HSIANG-FU KUNG1

1The Center for Emerging Infectious Diseases,The ChineseUniversity of Hong Kong, 2Department of Microbiology,The University of Hong Kong, 3Department of Chemistry,TheUniversity of Hong Kong

Purpose To test whether long-term expressed interferon-α in theliver (local) displays better anti-HBV effects that may overcome theshortcomes of interferon (strong side effects, short half-life, low localconcentration).Methods We made recombinant AAV-EGFP and AAV- Interferon-α1, and tested their anti-HBV activities in stable HBV-producing cellsand mice liver using hydrodynamic transfection mice model.Results We showed that rAAV vectors could effectively infect hepa-tocytes and stably express target gene interferon-α1, resulting in theinhibition of viral gene expression and viral replication. Both intra-cellular and extracellular HBsAg levels were significantly reduced invitro for at least two weeks. One week after systemic delivery of AAVvectors, mice were hydrodynamic transfected with a HBV producingplasmid. AAV vectors were successfully delivered to the liver, andinterferon-α1 was highly expressed in the hepatocytes. In the AAV-EPFG control group, high level HBsAg and viral DNA were detected.HBsAg was reduced to undetectable level and viral DNA was reducedhundreds folds in the AAV-IFN-α1 group.Conclusions AAV-mediated long-term expression of interferon-α1displayed potent inhibition of HBV gene expression and viral repro-duction.These results suggest that gene therapy may greatly improvethe anti-HBV efficacy of interferon-α and reduce its side-effects,making it an attractive treatment option.

#074

The frequency of Interleukin-2, Interleukin-6 aninterferon g-genes polymorphisms in hepatitis Bpatients in IranLEILA NAJAFI,1 ZAHRA HAJEBRAHIMI,1,2

LEILA ALIDOUST MASOULEH,1

MOHAMMAD HOSSEIN SOMI,1

MARYAM ZAFARGHANDI,1 MARYAM FIROOZI,1

MOHAMMAD REZA ZALI1

1Research Center for Gastroentrology and Liver Diseases,2Tarbiat Modarress university, Faculty of Science, GeneticsDepartment

Background Cytokines play an important role in the defenseagainst viral infection. Cytokines such as IL-2, IFN-g and TNF-alphahave been identified as participating in the viral clearance and the hostimmune response to HBV.The aim of the present study was to inves-tigate whether the IL-6(-174), IL-2(-330), IFN-g(-874) promoterpolymorphisms was associated with outcomes of HBV infection inIranian patients.Methods We examined 96 unrelated patients with Chronic Hepati-tis B and 96 spontaneously recovered, 96 control groups. The poly-morphisms were detected by PCR-RFLP.Results The IL-2, IL-6 and IFN-g genotype distribution and allelefrequency are shown in Table. There is no association between eachpolymorphisms and chronic hepatitis B patients over spontaneouslyrecovered (p > 0.05).

Table Distribution of polymorphisms and alleles frequency in ourstudied groups

Polymorphisms Healthy Spontaneous Chronic Controls recovered HBV HBVN (%) N (%) N (%)

IL-2(-330) T/T 27.4% 27.1% 24%G/T or 72.6% 72.9% 76%G/G G = 47.8% G = 47.3% G = 49%AlleleFrequency

IL-6(-174) G/G 47.4% 58.2% 53.6%C/G or 52.6% 41.8% 46.4%C/C C = 28.2% C = 26.5% C = 27%AlleleFrequency

IFN-g(-874) T/T 26.1% 35.3% 14.5%A/T or 73.9% 64.7% 85.5%A/A A = 60% A = 43% A = 52%AlleleFrequency

#075

Immunization of a recombinant plasmid encodinghepatitis C virus core proteinFATEMEH KHATAMI,1 ALI KARAMI,2

MOHAMMAD NABI SARBOLOKI,3

MOHAMMAD HOSSEIN SANATI2

1Research center for gastroentrology and liver diseases, 2NationalResearch Center for Genetic Engineering and Biotechnology,Tehran, I.R. Iran., 3Institute of Biophysics and Biochemistry,Tehran university, Iran

Introduction The HCV is the major cause of nonA nonB viralhepatitis. In order to increase the potency of DNA vaccines, conven-tional adjuvants were used to find the best vaccine.Methods HCV cDNA was isolated from an Iranian individual withchronic hepatitis C. Plasmid DNA was subsequently purified in freeendotoxin method. Four adjuvants were used: aluminium phosphate,CpG motif, dendrosome and CpG + alum. BALB/c female mice wereimmunized by 100 microlitre of different immunogen. The antibodyresponse was determined by using HCV core Elisa kit.Result Dendrosome improve the antibody response especially afterthird injection. Mixture of alum and CpG with HCV core pcDNA3stimulated the anti-core antibody response. It has been used toincrease the efficiency of HCV core pcDNA3 immunization that wassuccessful.Discussion Our data indicate the anti-HCV core humoral immuneresponse generated following DNA immunization could be modifiedby dose of plasmid, times of injection and kind of adjuvant.

A98 Journal of Gastroenterology and Hepatology (2006) 21 (Suppl. 2)

#076

Highly endemic hepatitis B infection in Rural VietnamVAN THI-THUY NGUYEN,1

MARY-LOUISE MCLAWS,1 GREGORY J DORE2

1School of Public Health,The University of New South Wales,2Viral Hepatitis Research Program, NCHECR, University ofNew South Wales

AbstractBackground Hepatitis B is a major public health problem inVietnam. However, estimates of the prevalence of hepatitis B virus(HBV) and hepatitis Delta virus (HDV), and risk factors in ruralVietnam are limited.Methods A cross-sectional seroprevalence study was undertaken intwo rural districts in Thai Binh province. The study population wasrandomly selected using multi-stage sampling. Demographic andbehavioral risk information and serological samples were obtainedfrom 837 participants.Results Mean age was 42.3 years ± 15.8 (range, 16–82 years) and50.8% were female. Prevalence of HBcAb and HBsAg was 68.2% and19.0%, respectively, and eAg was detected in 16.4% of the HBsAgpositive group. Prevalence of HDV was 1.3% in the HBsAg positivegroup. Factors associated with HBV infection (HBcAb or HBsAg pos-itive) were age sixty years or older (OR 3.82, 1.35–10.80; p = 0.01),residence in Vu Thu district (OR 3.00, 2.16–4.17; p < 0.0001), hos-pital admission (OR 2.34, 1.33–4.13; p = 0.003) and history ofacupuncture (OR 2.01, 1.29–3.13; p = 0.002). Household contactwith a person with liver disease (OR 2.13, 1.29–3.52; p = 0.003),reuse of syringes (OR 1.81, 1.25–2.62; p = 0.002) and sharing ofrazors (OR 1.69, 1.03–2.79; p = 0.04) were independent predictorsof HBsAg positivity. Alanine aminotransferase (ALT) level was ele-vated (>40U/L) in 43% of the HBsAg positive group.Conclusion HBV infection and HBV related liver disease remainsa serious public health problem in rural Vietnam. Poor infectioncontrol activities in health care settings partly accounted for the highHBV prevalence in this region. Universal HBV infant vaccination andimproved infection control procedures are required for improvedHBV control in Vietnam.

#077

Dose-response relation of interferon-alpha in patientswith HBeAg positive chronic hepatitis B:meta-analysis and meta-regression of randomizedtrialsXIN SUN,1,2 WENXIA QIN,1 RONGLE ZHOU,3

YOUPING LI1

1Chinese Evidence-Based Medicine Center,West China Hospital,Sichuan University, 2Department of Clinical Pharmacy,WestSchool of Pharmcy, Sichuan University, 3West China MedicalSchool, Sichuan University

This study examined dose-response effect of interferon-α in HBeAg-positive CHB patients. We searched 5 electronic databases, allupdated to September 2005. Randomized trials of interferon-α vs.non-antivirals were eligible. Quality of trials was assessed by standardinstrument. Meta-regression and meta-analysis were used to examinethe dose-response relation. Thirty-three trials were included (n =2164). Dose of interferon-α ranged from 1–10 MU, and durationfrom 4–24 weeks. Eighteen trials were medium-to-high in quality.HBeAg clearance was responsive to dose (coef = 0.156, 95%CI =0.028–0.28) and duration (coef = 0.076, 95%CI = 0.0048–0.15), butnot for HBV-DNA clearance. Stratified analyses showed that ≥5 MUand 16–24 week interferon-α could effectively clear HBeAg (OR =3.28, 95%CI = 2.31–4.66; OR = 3.28, 95%CI = 2.16–5.00), andHBV-DNA (OR = 2.80, 95%CI = 2.03–3.86; OR = 2.58, 95%CI =1.62–4.12). HBeAg seroconversion could be seen in all-dose groups(OR = 2.02, 95%CI = 1.37–2.97). In conclusion, HBeAg clearancedose-responsive. A ≥5 MU and 16–24 week interferon-α could effec-tively clear virological and serological markers.

0

0.5

1

1.5

2

2.5

Day

OD

in 4

50n

m

10 microgram HCV corepcDNA3+PBS



10 microgram HCV corepcDNA3+CpG motif



10 microgram HCV corepcDNA3+CpG motif+Alum

25 microgram HCV corepcDNA3+CpG mitif+Alum

50 microgram HCV corepcDNA3+CpG motif+Alum

10 microgram HCV corepcDNA3+Dendrosome

25 microgram HCV corepcDNA3+Dendrosome

1 17 42 62

Table Immunization of HCV core pcDNA3 with four different adjuvants

Figure 1. The dose (left) and duration (right) of interferon-αin relation to the HBeAg clearance (log scale). Y-axis means thelog scale of HBeAg clearance, X-axis means the dose of inter-feron-α. Each trial is represented by a circle, the area of whichrepresents the trial’s precision. Larger circles represent trial that contribute more information. Regression equation: logOR(HBeAg clearance) = 0.34 + 0.156 × dose, and log OR(HBeAg clearance) = −0.51 + 0.076 × duration

#078

The rheumatic manifestations in patients with chronic hepatitis C virus infection and mixed cryoglobulinaemiaOLGA VOROZHBYT,1 SERGE CHOOKLIN1

1Medical University, Lviv, Ukraine

Background Chronic hepatitis C virus infection is associated withmany extrahepatic manifestations. The prevalence of rheumatologicsymptoms in patients with mixed cryoglobulinemia associated tochronic hepatitis C virus (HCV) infection was investigated in ourresearch.Methods One hundred thirty patients with chronic HCV infectionand cryoglobulinemia were recruited. The presence, concentration,and type of cryoglobulins were tested by immunofixation. Rheuma-toid factor (RF) and antinuclear antibody (ANA) were also measured.


HCV genotype was determined by PCR with genotype specificprimer.Results Type II cryoglobulinemia was identified in 42 cases and itwas of type III in 15. HCV-RNA genotype was determined in 130patients: 109 (83.8%) were infected by genotype 1b, 14 (10.7%) bygenotype 3a and 7 patients (5.4%) was the mixed genotype. RF andANA were, respectively, present in 110 (84.2%) and 62 (47.4%)patients at low titer (RF < 50UI/ml, ANA = 1 :80). Of the 130patients, 57 (43.8%) complained for rheumatic symptoms.The arthri-tis was usually symmetric and involved the small joints of the hand,wrist, and feet, but may involve larger joints, such as the elbows,ankles, and knees.The mean cryocrit value in these patients was 2,6%,while in patients with HCV infection, liver disease, and cryoglobu-linemia without rheumatic symptoms the cryocrit value was lowerthan 0.5% in 50% of cases.Conclusion It is concluded that patients with chronic HCV infec-tion reported a wide variety of rheumatic manifestations, impairingtheir quality of life, with discrete frequency.

#079

Peripheral blood dendritic cell subsets and seruminterleukin-12 levels in hepatitis C virus-infectedpatients with persistently normal and elevated alanineaminotransferase levelsHODA EL AGGAN,1 NAHLA FARAHAT,2

FATHALLAH SIDKEY,1 EMAN EMAM3

1Department of Medicine (Hepatobiliary Unit), 2Department ofClinical Pathology, 3Department of Pathology, Faculty ofMedicine, University of Alexandria, Egypt

Hepatitis C virus (HCV)-specific T-cell immune responses appear toinfluence the outcome of infection and require activation by dendriticcells (DC). The present work was designed to assess DC subsets inperipheral blood and serum levels of interleukin-12 (IL-12) inpatients with chronic hepatitis C in relation to disease activity.Twentyeight patients with chronic hepatitis C [15 with elevated serumalanine aminotransferase (ALT) levels and 13 with persistentlynormal ALT] and 12 healthy subjects were included in the study.Peripheral blood myeloid and lymphoid DC subsets were assayedusing 3-color flow cytometry and were differentiated by the expres-sion of CD11c or CD123 on the cell surface respectively. Determi-nation of IL-12p70 heterodimer in serum was performed usingenzyme-linked immunosorbent assay. The percentages of peripheralblood DC subsets (CD11c+ and CD123+), the CD11c+ DC/CD123+

DC ratio and the serum IL-12 levels were significantly lower inchronic hepatitis C patients than in healthy subjects and in patientswith elevated ALT than in those with normal ALT (P < 0.05). Thereduction in circulating DC subsets and serum IL-12 levels showednegative correlations with serum levels of ALT and the histopatho-logical grading and staging scores (P < 0.05). In conclusion, the defi-ciencies in circulating DC subsets and IL-12 production may play akey role in the progression of liver disease in chronic HCV infectionand may provide a potential new goal for HCV immunotherapy.

#080

The efficacy of thymosin-apha in the treatment of chronic hepatitis B: a meta-analysis of randomized trialsXIN SUN,1 WENXIA QIN,1 RONGLE ZHOU,1

YOUPING LI1

1Chinese Evidence-Based Medcine Center,West China Hospital,Sichuan University

A meta-analysis of randomized trials was conducted to study the effi-cacy of thymosin-α in patients with HBeAg-positive chronic hepati-

tis B (CHB). We searched 5 electronic databases, all updated to Sep2005. Randomized trials of thymosin vs. non-antiviral interventionsfor HBeAg-positive CHB patients were eligible. Publication bias wasdetected by Egger’s regression. Heterogeneity was examined by Q-statistics. Meta-regression was performed by regimens and ethnics.Totally, seven trials were included (n = 762). Three trials were highin quality. Regimens ranged from 1.6-mg subcutaneous injection for52 weeks to 200-mg iv.gtt for 12 weeks. It was found that thymosin-α could effectively clear HBeAg (OR = 2.87, 95%CI = 1.90–4.35)and HBV-DNA (OR = 1.79, 95%CI = 1.00–3.22), and achieve sig-nificant HBeAg seroconversion (OR = 4.55, 95%CI = 1.90–4.35). Butit could not effectively normalize aminotransferase (OR = 0.99,95%CI = 0.73–1.36). In conclusion, thymosin-α might be effective inclearing virological and serological markers. However, more qualitytrials are needed to confirm the efficacy and identify the best regimen.

#081

Comparative effectiveness of lamivudine vs. adefovirin patients with HBeAg-positive chronic hepatitis B:combining direct and indirect evidenceXIN SUN,1 RONGLE ZHOU,1,2 WENXIA QIN,1

YOUPING LI1

1Chinese Evidence-Based Medicine Center,West China Hospital,Sichuan University, 2West China Medical School, SichuanUniversity

Few evidence compared lamivudine with adefovir in HBeAg-positiveCHB patients. In this study, we studied their comparative effective-ness by direct and indirect evidence. We searched 5 electronic databases, all updated to Sep 2005. We conducted meta-analysis ofrandomized trials comparing lamivudine vs adefovir in patients withHBeAg-positive CHB to obtain estimates. An indirect adjusted com-parison of lamivudine vs adefovir was performed by using summarystatistics of lamivudine vs non-antivirals and adefovir vs non-antivirals, which were obtained by meta-analysis of randomized trials.Totally, 10 trials were included. Of them, 1 compared lamivudine vsadefovir (n = 38), 9 compared 100-mg lamivudine vs placebo/untreated (n = 1097), and 1 compared adefovir vs placebo/untreated(n = 512).The direct and indirect summary statistics were calculated(Table 1). Both direct and indirect comparison showed that adefovir

Table 1 The direct and indirect comparison of lamivudine vs adefovir

Lamivudine vs adefovir No. of No. of Odds 95% Cltrials subjects ratio

Loss of HBeAgDirect comparison 1 38 0.16 0.01–3.00Indirect comparison 6 1083 1.00 0.57–1.73

Lamivudine vs 5 571 2.42 1.70–3.46placebo/untreated

Adefovir vs placebo/ 1 512 2.43 1.59–3.71untreated

HBeAg seroconversionDirect comparison 1 38 0.20 0.009–4.44Indirect comparison 6 1031 1.25 0.51–3.06



Normalization of ALTDirect comparison 1 38 0.11 0.013–0.97Indirect comparison 8 1151 0.47 0.32–0.70



*clearance of HBV-DNA was not compared as the trial comparing ade-fovir vs placebo used a differing measure on HBV-DNA.


and lamivudine were statistically equivalent in loss of HBeAg andHBeAg seroconversion, and adefovir was superior in normalization ofALT. Indirect comparison narrowed the confidence intervals.

#082

Prevalence, risk factors and intrafamilial spreading ofHepatitis B Virus in Gonbad, IranAKRAM POURSHAMS,1 REZA MALEKZADEH,1

JAFAR NASIRI2

1Tehran University of Medical Sciences, 2Shahrekord Universityof Medical Sciences

Aim HBV infection in a general population.Methods 1343 adult inhabitants of three villages and city ofGonbad in north-eastern Iran were invited to the study. Risk factorsfor HBV infection were accessed through interview and HBs Ag waschecked for participants. HBs Ag positive subjects underwent physi-cal examination by a gastroenterologist and serum ALT measure-ment. All family members who lived with a HBs Ag positive subjectin a home and were older than 2 years underwent HBs Ag and HBcAb checking.Results 1035 (mean age 51.3 ± 11.7 years) participated to thestudy and 44 out of them (4.25%) were HBs Ag positive. There wasno difference for unsafe sexual and blood contacts between HBs Agpositive and HBs Ag negative subjects. None of the HBs Ag positivesubjects had elevated serum ALT level or a liver failure sign. Seventy(6%) and 20(7%) out of the 279 family members were positive forHBs Ag and HBc Ab respectively. The most frequent relative of HBsAg positive cases was daughters (64%). All HBs Ag positive familymembers were older than 12 years.Conclusions Prevalence of HBs Ag positive in Gonbad is muchmore than average rate of Iran. Intrafamilial spread is in medium rageand daughters are at more risk than other family members. NeonatalHBV vaccination program, which is started 12 years ago is an effi-cient prevention method.

#083

Relationship between seroprevalence of anti-HAV andage: Impact on screening before vaccinationCHUN-TAO WAI,1 BELINDA MAK,1

AUNG MYAT OO,1 SENG-GEE LIM1

1Yong Loo Lin School of Medicine, National University ofSingapore

Introduction Acute hepatitis A infection is a vaccine-preventabledisease. In endemic areas like Southeast Asia, it is unknown if testingof anti-HAV should be done prior to hepatitis A vaccination.Aim To evaluate prevalence of anti-HAV IgG in the general popu-lation, to evaluate the effectiveness of prevaccination testing.Methods Free and voluntary testing of anti-HAV IgG was per-formed in conjunction to a public medical education event. Volun-teers were asked about history of past hepatitis infection andvaccination history, and tested for hepatitis A markers. Impact of pre-vaccination tested was then evaluated.Results Three hundred and eighty-two volunteers were tested. Agewas 50.7 ± 0.7 (median 52, range 12–84) years. Twenty-five (6.5%)had prior hepatitis A vaccination, and 5 (1%) had prior hepatitis Ainfection. 220/358 (58%) were positive for anti-HAV IgG. At multi-variate analysis, age is the only significant factor associated with pos-itive anti-HAV IgG. Area under ROC curve of using age to predictanti-HAV IgG positivity was 0.75 (95% 0.70–0.80). Two age cutoffs,40 and 55 years, were selected from the ROC curve. When age <40years, only 13/70 (19%) were positive for anti-HAV IgG. When age>55 years, 116/144 (81%) were positive for anti-HAV IgG.

Conclusion In Singapore, prior to vaccination, patients <40 yearsare unlikely to be positive for anti-HAV IgG and they could be vac-cinated without prevaccination testing. In contrast, patients aged >55years should be screened for anti-HAV IgG before vaccination.

#084

Effects of psycho-educational intervention on lifequality of patients with chronic liver diseasesFARKHONDEH SHARIF,1 SADROLLAH MOHEBI,1

MEHDI SABERFIROUZI,1

HAMID REZA TABATABAEE,1

MALIHEH GHOLAMZADEH1

1shiraz university of medical sciences

Background Chronic liver diseases (CLDs) is a progressive disor-der which has a significant impact on the well-being of patients andleads to significant morbidity. CLDs are characterized by distur-bances in physical and psycho-social aspects of well-being. Psycho-educational interventions that target functional factors could bebeneficial for patients with CLDs.Methods An interventional study was conducted on 110 patientswith CLDs in Shiraz Liver Transplantation Center. Subjects with therequired CLDs criteria were selected and randomly divided intoexperimental (55) and control (55) groups. A two part questionnairewith 25 items concerning demographic and general information and29 items regarding life quality was used for measuring life quality.Thepsych-educational needs of the experimental group were assessed ina session before intervention, then they took part in three sessionsindividually and one session in group. The questionnaires were filledin again for both groups one day and three months after interventionbut the control group did not receive intervention program.Results Finding revealed no significant differences between the twogroups from the point of demographic characteristics such as maritalstatus, gender, etc . . . (p > 0.05) and from the point of Para-clinicalvariables, no statistically significant differences were found (p > 0.05).Findings revealed statistically significant differences in all domain oflife quality in the experimental group three months after the inter-vention (p = 0.001), while there were no statistically significant dif-ferences in the control group.Conclusion Results of this study revealed that psycho-educationalintervention had a significant effect on life quality of patients withCLDs.

#085

Effect of lamivudine combination with interferonalpha on growth factors in respect to inflammationand fibrosis patients in chronic hepatitis BOLEKSII KORZH,1 ELIZAVETA LAVROVA1

1Kharkov Medical Academy of Postgraduate Education

Growth factors (GF) that participate in regeneration and apoptosishave an important role in chronic liver diseases. We analyzed serumGF concentration during combining treatment with lamivudine(LAM) and interferon alpha (IFNalpha), and correlated it with mor-phological liver failure in chronic hepatitis B. The levels of GF weredetermined in sera by ELISA method at baseline, 12, 24 and 48 weekof therapy in 46 patients treated with LAM and IFNalpha and in 23healthy subjects. Blind liver biopsies were done before treatment withhistological grading and staging examination.The hepatocyte growthfactor (HGF) and epidermal growth factor (EGF) were markedly ele-vated prior the treatment and decreased during the therapy, althoughthey did not reach the normal level. In non-responding (NR) patients,HGF and EGF were higher than that in responders (R), however dif-ferences were not significant. Before the treatment thrombopoietin(TPO) level was significantly lower in R than in NR (P < 0.05).


Platelet-derived growth factor (PDGF) concentration was lower inchronic hepatitis B than in healthy subjects and decreased during thetreatment. A significant positive correlation was observed betweeninflammatory activity in the liver tissue and the concentration ofHGF, TPO, and a significant negative correlation between this activ-ity and EGF and PDGF. Serum HGF concentration was higher inmore advanced fibrosis (P < 0.05). The decrease in PDGF can be aneffective prognostic marker of the treatment and HBV elimination.Decreasing HGF, EGF, and PDGF can influence the inhibition ofinflammatory and fibrotic processes in the liver during the antiviraltreatment.

#086

Cryoglobulinemia and hepatic fibrosis in chronichepatitis CANASTASIOS MIHAS,1,2 MITCHELL SHIFFMAN,1,2

ROBERT LIPPMAN,2 DOUGLAS HEUMAN1,2

1Virginia Commonwealth University Medical Center, 2McGuireVAMC

Background The relationship of cryoglobulinemia to severity ofliver disease and its importance as a cause of renal dysfunction inchronic hepatitis C remain unclear.Aims To determine the relationship between cryoglobulinemia andhepatic fibrosis and the effects of cryoglobulinemia on renal functionin chronic hepatitis C.Methods 153 consecutive patients undergoing initial liver biopsyfor chronic hepatitis C were included in this study. All patients hadliver imaging by US as well as routine hematologic, biochemical andserological testing by accepted standard laboratory methods. Severityof hepatic fibrosis was assessed by consensus of two reviewers usingthe 6-stage Ishak scale.Results Serum cryoglobulins were detected in the sera of 76 (49%)patients. These patients exhibited a trend toward more advancedfibrosis than the CG-negative group (mean Ishak score 2.56 ± 1.8 vs2.27 ± 1.7), and the prevalence of liver cirrhosis in the CG-positivegroup was higher than in the CG-negative group (15% vs 7.5%).While serum creatinine levels did not differ between CG-positive andCG-negative groups (1.09 ± 0.64 vs 1.05 ± 0.35), renal insufficiency(serum creatinine > 1.4) in the CG-positive group was more commonthan in the CG-negative group (7.9% vs 2.6%).Conclusions 1. Almost half of our patients with chronic HCVinfection had detectable serum CG. 2.While most HCV patients withCG are noncirrhotic and have normal renal function, cryoglobuline-mia is associated with a small increase in prevalence of cirrhosis andrenal insufficiency.

#087

Influence of lamivudine combination with interferonalpha on the expression of Th1/Th2 type cytokines inpatients with hepatitis BOLEKSII KORZH,1 SERGIY KRASNOKUTSKIY,1

OLENA PAVLOVA1

1Kharkov Medical Academy of Postgraduate Education

The aim of this study was to investigate the relationship between theexpression of Th1/Th2 type cytokines and the effect of combinationtherapy with lamivudine (LAM) and interferon alpha (IFNalpha).Methods Th1/Th2 type cytokines were detected by ELISA andRT-PCR in 37 patients with chronic hepatitis B who were treated withLAM and IFNalpha for 36 weeks.Results The levels of IFN-gamma in the supernatant of PBMCcultures of the patients with hepatitis B were slightly lower than thoseof the controls. However, the levels of IL-4 were higher in the patientsthan in the controls. Cytokines measurements during LAM and

IFNalpha treatment showed a trend toward decreased levels of IL-4at 4, 12 and 36 weeks. However, the levels of IFN-gamma wereslightly increased following treatment. In patients with completeresponse to LAM combination with IFNalpha, the levels of IFN-gamma were higher than that of pre-treatment at 36 weeks followingtreatment, and the levels of IL-4 decreased obviously at 12 and 36 weeks. The mRNA levels of Th1/Th2 type cytokines were posi-tively correlated with the levels of supernatant.Conclusions (1) The expression of Th2 type cytokines is predom-inant in patients with chronic hepatitis B. (2) The effect of lamivu-dine combination with interferon alpha therapy may play a role inmodulating the balance of Th1/Th2 type cytokines. (3) Variations ofthe levels of Th1/Th2 type cytokines could be used as a predictor ofclinical response during combining treatment with lamivudine andinterferon alpha.

#088

Correlation between endothelial haemostatic markersin patients with chronic hepatitis CIRYNA KORZH,1 INGA FEDOTOVA1

1Kharkov Medical University

We hypothesized there may exist a correlation between the plasmalevels of von Willebrand factor, thrombomodulin, and tissue plas-minogen activator antigen (tPAag) as markers of endothelial cell dys-function and the serum concentrations of soluble adhesion moleculesand monocyte chemoattractant protein-1 (MCP-1) in patients withchronic hepatitis C. Patients (n = 117) with chronic hepatitis Cwithout malignancy, a history of venous thrombosis, orantiviral/immunosuppressive therapy within the last six months andmatched controls (n = 115) were included.To evaluate possible influ-ence of acute phase reaction, reinvestigation was performed after 6 months. The concentrations of von Willebrand factor, thrombo-modulin and tPAag in plasma were significantly higher in patientswith chronic hepatitis C as compared to healthy subjects (p = 0.017,p < 0.05 and p < 0.001, respectively), still statistically significant after6 months and also after adjustment for established risk factors. Thepatients also had significantly higher concentrations of soluble vascu-lar cell adhesion molecule-1 (sVCAM-1) and MCP-1 compared tohealthy controls (p < 0.001 for both comparisons).There were strongcorrelations between the concentration of soluble intercellular adhe-sion molecule-1 (sICAM-1) and von Willebrand factor in patientswith chronic hepatitis C (r = 0.64, p < 0.001), between the concen-tration of thrombomodulin and sVCAM-1 (r = 0.62, p < 0.001), andbetween tPAag sVCAM-1 (r = 0.51, p < 0.05).The strong correlationbetween markers of endothelial dysfunction and soluble adhesionmolecules in patients with chronic hepatitis C strengthen the viewthat an ongoing stress on endothelial cells is present in these patients.

#089

Silymarin prevents hepatocytes from palmitateinduced cell death and Interleukin-8 productionZHENYUAN SONG,1 MING SONG,1

SILVIA URIARTE,1 ION DEACIUC,1 DAVID LEE,2

CRAIG MCCLAIN1

1University of Louisville, 2Harvard Medical School

Emerging evidence suggests that elevated serum FFA levels play anetiologic role in the pathogenesis of nonalcoholic steatohepatitis(NASH), and this has been postulated to be a critical link betweenobesity, insulin resistance and the risk of NASH. Silymarin is aflavonoid, extracted from the milk thistle Silibum marianum. Althoughthe hepatoprotective properties of silymarin have been reported bothfrom in vitro and in vivo studies, its possible role in preventing lipo-toxicity from FFA, specifically saturated FFA such as palmitate, has


never been investigated. Here we investigated the effects of supple-mentation of silymarin on palmitate induced cell death and cytokine(IL-8) production in hepatocytes. HepG2 cells, a human hepatomacell line, were treated with palmitate in the absence or presence ofsilymarin. Supernatants or cell lysates were collected at different timepoints. Cell death was measured by DNA fragmentation ELISA andcaspase-3 activity assay. IL-8 production was assayed by measuringIL-8 release in the supernatants using an ELISA kit. We have foundthat pretreatment with silymarin protected HepG2 cells from palmi-tate induced cell death and the protection was independent on itsantioxidant character. Moreover, palmitate caused increased IL-8production from HepG2 cells and silymarin supplementation pre-vented the enhancement of IL-8 production by palmitate. Althoughcaspase-3 inhibitor also attenuated palmitate induced cell death, it didnot prevent palmitate induced IL-8 production, suggesting that pre-vention of IL-8 production by silymarin was cell death independent.

#090

Applying sequencing to identify hepatitis B virusgenotypes and mutationsTAN DAT HO,1 THU THUY PHAM THI,1

BAO TOAN NGUYEN,1 THANH TONG NGUYEN1

1MEDIC MEDICAL CENTER

Hepatitis B Virus can be classified into 8 genotypes A through Hbased on an intergroup divergence of 8% or more in the completenucleotide sequence. We have applied the Sequencing by TruGene(Bayer) to identify Hepatitis B Virus Genotypes and Mutations. FromAugust 2004 to November 2005, in MEDIC we have implementedsequencing for 69 cases Vietnamese with Chronic Hepatitis B onLamivudine treatment in which: 55 males (79.7%) and 14 females(20.3%), medium age: 34.6 ± 10.3. All cases are HBVDNA (+) inwhich: 50 cases are HBeAg (+) (72.5%) and 19 cases HBeAg (−)(27.5%). There are only two genotypes: genotype B(65.2%) andC(34.8%)Totally there are 41 cases (59.4%) mutations with following estab-lished proportion and types: L180M (5.8%); L180M & M204I(8.7%); L180M & M204V (30.4%); L180M, M204V & V207I(1.4%); M204I (8.7%); M204I & V207I (1.4%); M204V (2.9%).There are 25 cases mutations in 45 cases of genotypes B (55.6%) and16 cases in 24 cases of genotypes C (66.7%). All patients are onLamivudine treatment with average time is 2.4 ± 0.9 years, the cor-responding number of patients in treatment period of 1, 2, 3, 4 yearare 10, 29, 21, 9. The proportion of developing mutations after 1, 2,3, 4 years of treatment with Lamivudine is 20%; 55.2%; 76.2%;77.8%.We have found that the sequencing plays a very important role inidentifying Hepatitis B Virus Genotypes and Mutations. From thoseresults we can approach an optimal treatment course.

#091

Induction of type I interferon in human hepatocytes is dependent on protein kinase PKR and retinoic acid-inducible gene I (RIG-I)HAIZHEN ZHU,1 HUIJIA DONG,1 JOHN ELYAR,1

MICHAEL BUTERA,1 DAVID NELSON,2 CHEN LIU1

1Department of Pathology, Immunology and LaboratoryMedicine University of Florida, 2Department of MedicineUniversity of Florida

Induction of Type I Interferon in Human Hepatocytes is Dependenton Protein Kinase PKR and Retinoic Acid-Inducible Gene I(RIG-I)Haizhen Zhu, Huijia Dong, John Elyar, Michael Butera, David Nelsonand Chen LiuDepartment of Pathology, Immunology and Laboratory Medicine, Univer-sity of Florida, Gainesville, FL32610

Introduction Production of type I IFN has been extensivelystudied and shown to be induced by dsRNA. Most of recent studieswere conducted in human 293 or epithelial cells. The molecularmechanism of these events in hepatocytes is little known.Methods Poly I:C was added to the Huh7 cell culture medium ortransfected with lipofectin into the cells. IFN-beta and IL-28A RNAwere assayed by real-time PCR. Protein PKR or IRF3 was determinedby Western blot.Results Huh7 cells do not express IFN-beta and IL-28A RNA inresponse to extracellular poly I:C. Intracellular poly I:C induced theproduction of IFN-β and IL-28A mRNA in Huh7 cells. Chloroquinecannot attenuate IFN-β responses to poly I:C. Poly I:C responsive-ness could not be conferred on Huh7 cells by expression of TLR3.Either 2-aminopurine or PKR siRNA partially ablated intracellularpoly I:C-induced IFN-β mRNA production. RIG-I siRNA also par-tially inhibits the expression of type I IFN in Huh7. Treatment withPKR siRNA and RIG-I siRNA together completely block the IFN-βexpression in Huh7.Conclusion Type I IFN response to intracellular dsRNA in humanhepatocytes is through PKR and RIG-I signaling pathways but TLR3-independent.

#092

Infectious dose of HBV in terms of copy number ofDNA in human seraCHU CHIEH HSIA,*1 ROBERT H PURCELL,2

MAHMOOD FARSHID,1 PETER A LACHENBRUCH,1

MEI-YING W YU1

1Center for Biologics Evaluation and Research, Bethesda, MD,USA, 2National Institute of Allergy and Infectious Diseases,NIH, Bethesda, MD, USA

Information about the minimum copy number of HBV genomesrequired for infecting a chimpanzee are not available. The informa-tion would be useful as a reference for antiviral drug and vaccinedevelopment.They are also useful for monitoring HBV patients undertreatment, as well as for regulating the blood screening and diagnos-tic tests for HBV DNA.Materials and Methods Sera from HBV carriers of the three mostcommon subtypes of HBV were studied. Their infectivity titers hadbeen evaluated previously in chimpanzees. The HBV DNA copynumbers in these sera were quantified using both TaqMan PCR anda nested PCR at limiting dilution.Results The genotype of HBV adw, ayw and adr inocula was A, D,and C, respectively as determined by type-specific amino acids in theS gene of HBV.The concentration of HBV DNA in the sera was deter-mined to be 5.3 × 109, 2.5 × 109, and 3.1 × 108 genome equivalents(gEq)/mL for sera containing adw, ayw, and adr HBV respectively.The chimpanzee infectivity titer (CID-50)/mL of these HBV-containing inocula was previously determined to be 107.5 for both adwand ayw, and 108 for adr.Conclusion The minimal copy number of HBV DNA that wasinfectious for the chimpanzee was estimated to be 166 gEq for adw(genotype A), 78 gEq for ayw (genotype D), and 3 gEq for adr (geno-type C). These data indicate that the relative infectivity of HBV canvary in different carriers and that the ratio of gEq to CID-50 can beas small as 3.

#093

Study on detection method of mutations in hepatitis Bvirus precore and core promoterJINGJUAN DING,1 YUEHUI LIU,1 MEI WANG1

1Guiyang medical college


DING-JINGJUAN,* LIU-YUEHUI, WANG-MEI*Department of infectious diseases, Guiyang medical college,China

Objects To establish a simple and accurate method for detectionmutations in HBV precore1896 and basic core promoter(BCP)1762/1764.Methods HBV precore, core promoter gene fragments were ampli-fied by mismatched polymerase chain reaction (mPCR). PCR prod-ucts were digested by Bsu36I, BclI and subjected to agarose-gelelectrophoresis, respectively. The partters of restriction fragmentlength polymorphism (RFLP) were disdinguished. 127 sera were ana-lyzed for the mutations in precore1896, BCP1762/1764 by mPCR-RFLP and direct sequencing. Two sera were also analyzed by clonesequencing.Results In 127 sera, 125 could be analyzed HBV precore 1896,BCP1762/1764 situation by mPCR-RFLP, 121 could be analyzed bysequencing. In 119 sera which could be analyzed by two methods, 16were precore A1896 mutation, 34 were BCPT1762/A1764 mutation,21 were precore A1896 with BCP T1762/A1764 mutations, 48 werewild strains. The results were completely compatible and there wasno significant difference in detective rate between two methods(98.42% vs 95.21%, p > 0.05). The sequence of five clones from oneserum identified precore mutation by mPCR-RFLP were all A1896mutant strains. Another serum identified mixture infection by mPCR-RFLP, one clone was A1896 mutant and four G1896 wild strains.The results of mPCR-RFLP were verified by cloning.Conclusions Comparision with sequencing, the mPCR-RFLPmethod is simple, accurate and can be used in large-scale surveys andclinical research.

#094

The comparison of hepatitis B e antigen status withclinical and histopathological status in chronichepatitis BJIE PENG,1 KANGXIAN LOU,1 JINLIN HOU1

1Department of Infectious Diseases, Nangfang Hospital,Nangfang Medical University, Guangzhou

Objective Both hepatitis B e antigen (HBeAg)-positive andHBeAg-negative chronic hepatitis B (CHB) are endemic in China,and the prevalence, clinical and histological features of HBeAg-negative group have not been fully studied.To elucidate those, a totalof 743 successive in-patients with biopsy-proved CHB were included.Methods We studied the correlation among alanine transaminase(ALT) levels, hepatitis B virus (HBV) DNA semiquantification, liverhistopathological data of 743 patients with CHB, divided into twogroups according to the HBeAg status.Results Of the 743 successive in-patients, 267 (35.9%) wereHBeAg-negative.The results showed that the HBeAg- negative grouphad significantly lower serologic HBV DNA levels (<100pg/mL in63.0% vs. 42.6% of HBeAg-positive group, P < 0.001), but moresevere inflammation (inflammatory scores of histological activityindex, HAIinf ≥9 in 58.1% vs. 46.0% of HBeAg-positive group, P <0.001) and more severe fibrosis (fibrosis scores of histological activ-ity index, HAIfib ≥3 in 45.3% vs. 27.9% of HBeAg-positive group, P< 0.001) of liver histology. In HBeAg-positive patients, increasingALT levels were associated with significantly higher inflammation andfibrosis scores and lower HBV DNA levels. However, it was not thecase in HBeAg-negative cases. There was an obverse correlationbetween HBV DNA levels and histology scores (HBV DNA >100pg/mL had HAIinf ≥9 in 91.9% and HAIfib ≥3 in 65.7%; while<20pg/mL had HAIinf scores ≥9 in 8.2% and HAIfib scores ≥3 in12.3%) in HBeAg- negative patients.

Conclusions On clinical and histological background, the chronicHBeAg-negative hepatitis B is a different subpopulation from theHBeAg-positive counterpart.Key words Chronic hepatitis B; Hepatitis B e antigen;Histopathology.

#095

Induction of hepatitis B virus-specific immuneresponses in vivo without liver damages: A novelimmunotherapeutic approach to treat chronic HBV infectionSK. MD. FAZLE AKBAR,1 SHINYA FURUKAWA,1

OSAMU YOSHIDA,1 SAKIKO KANNO,1

YOIICHI HIASA,1 NORIO HORIIKE,1

MORIKAZU ONJI1

1Third Department of Internal Medicine, Ehime UniversitySchool of Medicine

Background Induction of adequate levels of hepatitis B virus(HBV)-specific immune responses is critical for recovery fromchronic HBV infection. However, this is usually associated withdamages of hepatocytes. In this study, we have evaluated a novelapproach to induce HBV-specific immunity without liver damages.Materials and Methods To achieve this goal, we explored thenatural adjuvant properties of antigen-presenting dendritic cells(DCs).The study was conducted in both HBV transgenic mice (HBV-TM) and normal subjects those were immunized with hepatitis B vac-cines but did not respond to it (vaccine nonresponders). DCs, isolatedfrom mouse spleen and human peripheral blood, were cultured withhepatitis B surface surface antigen (HBsAg) to prepare HBsAg-pulsedDCs. After confirming that HBsAg-pulsed DCs were immunogenicand free of contamination, we injected HBsAg-pulsed DCs intraperi-toneally to HBV-TM or intradermally to human (1–3 times).Results Administration of HBsAg-pulsed murine spleen DCsinduced anti-HBs, HBsAg-specific T cells and proinflammatorycytokines in HBV-TM (N = 10). But, there were no serological orhistological evidences of liver damages in any HBV-TM. Administra-tion of HBsAg-pulsed human blood DCs induced anti-HBs in vaccinenonresponders (N = 7). Again, there were no features of liverdamages.Discussion and Conclusions HBsAg-pulsed DCs represent anovel approach to induce HBV-specific immune responses in murineHBV carrier as well as in human vaccine nonresponders withoutcausing liver damages. These studies inspire optimism that HBsAg-pulsed DCs represent a novel therapeutic vaccine for treatment ofpatients with chronic liver diseases.

#096

Virologial and clinical aspect of occult HBV infection in patient with chronic hepatitis C:a multicentre studyEVANGELISTA SAGNELLI,1 GUIDO PIAI,1

LUCIA CIMMINO,2

CAMILLO DEL VECCHIO BLANCO,3

VINCENZO MESSINA,4 FELICE PICCININO,5

NICOLA COPPOLA,5 CECILIA MARROCCO,5

MICHELE IMPARATO,5 RAFFAELLA PISAPIA,5

CATERINA SAGNELLI,5 PIETRO FILIPPINI5

1U.O. GAstroenterologia, A.O. San Sebastian Caserta,2Department of Gastroenterology, Federico II University Naples,3Department of Gastroenterology, Second University of Naples,4U.O. Infectious Diseases, A.O. San Sebastian Caserta,5Department of Public Medicine, Second University of Naples


Occult HBV infection was tested in 89 consecutive HBsAg negativepatients with chronic hepatitis C (CHC) at the time of their first liverbiopsy (LB). For each patient three compartments (plasma, fragmentof LB and 6 millions of PBMC) were explored for presence of HBV-DNA by PCR; three sets of specific primers for HBV core, surfaceand x region were used. Of the 89 enrolled patients, 45 were anti-HBs/anti-HBc negative (Group 1), 18 anti-HBs/anti-HBc positive(Group 2) and 26 anti-HBs negative/anti-HBc positive (Group 3).Clinical and virological characteristic of these patients are reportedin the Table.Conclusion Presence of isolated anti-HCV may be considered agood marker of occult HBV infection in patients with CHC, since81% of positive cases show HBV genome, as detected by PCR, in theliver and/or in PBMC; in these patients presence of HBV-DNA isassociated with severe fibrosis

(8.8% of 41, p < 0.01). Twenty-eight patients (32.5%) experienced ahepatic flare during the follow-up; this event occurred more fre-quently in the 17 patients showing HBV-DNA than in those 69 HBV-DNA negative (64.7% vs. 24.6%, p < 0.005).Of the 13 patients HBV-DNA positive at the first observation, 11 weretreated with HAART containing lamivudine and became HBV-DNAnegative during the follow-up: of these 11, four turned HBV-DNApositive and showed a hepatic flare during lamivudine treatment, andtwo after lamivudine was discontinuated. A hepatic flare underlamivudine treatment occurred also in 2 of the 4 patient in whomHBV become detectable during the follow-up.Of the 49 patients with no hepatitis viruses marker, a hepatic flarewas observed in 26.3% of the 19 receiving HAART with immunereconstitution inflammatory syndrome (IRIS), in 11.8% of the 17patients receiving HAART without IRIS and in none of the 13patients left untreated.The study suggests that HBV occult infection, relatively frequent inanti-HIV positive patients, is frequently involved in the pathogenesisof hepatic flares.

#098

Clinical course of chronic hepatitis in HCV/HIVcoinfected patients: the lesson from sequential liver biopsiesCATERINA SAGNELLI,1 CATERINA UBERTI-FOPPA,1

GIUSEPPE PASQUALE,2 NICOLA COPPOLA,2

ADDOLORATA MASIELLO,2 LUCA ALBARELLO,1

CLAUDIO DOGLIONI,1 FELICE PICCININO,2

ADRIANO LAZZARIN,1 EVANGELISTA SAGNELLI2

1Depts Infect. Dis and Pathology, IRCCS S. Raffaele Milan,2Department of Public Medicine, Second University of Naples

Of 326 patients with HCV/HIV coinfection who underwent a LiverBiopsy (LB) at SRH, 35 had a 2nd LB 1–9 yrs later (median 4)(Cases). As control group 31 HCV monoinfected patients wereselected (Naples) (Controls): the interval between LBs was 2–10(median 5 yrs); 21 patients had genotype 1, 6 genotype 2 and 4 geno-type 3.Of the 35 Cases, 21 had HCV genotype 3 (group G3), 10 genotype1 and 4 genotype 4 (group G1/4). The following differences betweenG3 and G1/4 were observed: fibrosis >1: 80.9 vs 28.6% (P < 0.01) inthe 1th and 72.2 vs 42.8% in the 2nd LB; steatosis >1: 57.4 vs 28.6%in the 1th and 52.4 vs 28.6% in the 2nd LB.No substantial differences in liver histology were observed betweenCases and Controls, both in the 1th and in the 2nd LB, indicating asimilar outcome.

Group 1 Group 2 Group 3

N° of cases 45 18 26

Age, yrs, median 48 (23–68) 47 (27–63) 51 (34–66)(range)

with HBV-DNA 5 (1*) 2 (0*) 7 (2*)in liver

with HBV-DNA 0 8 (1*) 11 (5*)in PBMC plus liver

with HBV-DNA 0 1 (1*) 3 (2*)in PBMC

with 2 or more 5 (11%) 11 (61.1%) 21 (80.8%)PCR pos in at least compartment

HAI ≥ 7:HBV-DNA pos 2/5 (40%) 6/11 (54.5%) 15/21 (71.4%)HBV-DNA neg 15/40 (37.5%) 4/7 (45.5%) 0/5

stagingc 5–6:HBV-DNA pos 0/5 2/11 (18.2%) 14a/21 (66.7%) HBV-DNA neg 12A/40 (30%) 3/7 (43.8%) 0/5

*n° of patients with HBV-DNA also in plasma; A: 3 cases with cirrhosis;B: 6 cases with cirrhosis; C: staging according lshak

#097

Impact of occult HBV infection in HIV patients naivefor anti-retroviral therapyPIETRO FILIPPINI,1 NICOLA COPPOLA,1

RAFFAELLA PISAPIA,1 CARLO SCOLASTICO,1

CECILIA MARROCCO,1

ANTONELLA ZACCARIELLO,1 CESARE NACCA,2

CATERINA SAGNELLI,1 GIULIO DE STEFANO,3

TERESA FERRARO,4 CARLO DE STEFANO,5

EVANGELISTA SAGNELLI1

1Department of Public Medicine, Second University of Naples,2U.O. Infect. Dis., A.O. San Sebastiano, Caserta, 3U.O. Infect.Dis., A.O. Matera, 4U.O. Infect. Dis., A.O. Catanzaro,5U.O. Infect. Dis., A.O. Potenza

We enrolled 115 consecutive anti HIV positive, HBsAg negativepatients, naïve for antiretroviral treatment of whom 86 were followedup at least 6 months (range 6–36 months).Of these 86 patients, plasma HBV DNA was detected in 17 (19.8%)by PCR: in 13 patients at the first observation and in 4 only duringfollow-up. HBV-DNA positivity was more frequently found inpatients with antibody to HBV (31.1% of 45) than in those without

Cases* Controls**

1st L.B. 2nd L.B. 1st L.B. 2nd L.B.

HAI 4.8 ± 4.6 3.3 ± 2.8 4.8 ± 3.4 3.8 ± 2.4Fibrosis 2.4 ± 1.7 2.7 ± 1.9 2.8 ± 1.9 3.1 ± 1.7Steatosis 1.8 ± 1.5 1.8 ± 1.6 1.1 ± 1.3 1.4 ± 1.4

*Cases: median age 35 years (range 21–44); 583 ± 288 CD4; 54.3%females; 77.1% drug addictsBefore the 2nd LB, 14 received HAART and IFN ± ribavirin, 8HAART, 9 IFN ± ribavirine and 4 were left untreated**Controls: median age 44 years (range 27–58); 22.6% females; 0% drugaddicts80.6% received IFN + ribavirine before the 2nd LB


#099

Swine are a principal source of hepatitis E virusinfection in eastern ChinaYINGJIE ZHENG,1 SHENGXIANG GE,2 JUN ZHANG,2

QINGSHUN GUO,2 MUNHON NG,2 FADI WANG,3

NINGSHAO XIA,2 QINGWU JIANG1

1Department of Epidemiology, School of Public Health, FudanUniversity, Shanghai 200032, China, 2Centre for Research onMedical Molecular Virology of Fujian Province, XiamenUniversity, Xiamen, China, 3Centre for Disease Prevention andControl, Deqing County, Huzhou City, Zhejiang 313200, China

To ascertain whether swine are a source of human hepatitis E virusinfection, our study was conducted in two pig farming districts ofeastern China to determine the occurrence of the virus in swine andhumans, analyze phylogenetic relationship between concurrent swineand human isolates and assess risk of HEV infection attributable toswine farming.The results show that the detection rate of HEV RNAwas separately 68.8%(11/16) achieved for serum samples fromhepatitis E patients, 0.3% for healthy population with reactive IgManti-HEV, 9.6% (27/282) for swine feces and 3.1% (5/160) for swinebile.The viruses isolated from swine are consisted exclusively of geno-type IV and the isolates are phylogenetically related to most of thoseconcurrently isolated from humans. Those who are engaged in occu-pations related to swine farming sustain higher risk of HEV infection(75.9%, 258/340) than those who are engaged in other occupationand reside upstream of swine farming district (213/425, 50.1%) withan adjusted OR = 1.74 (95% CI = 1.24∼2.44) and the risk increaseswith duration of the occupational exposure. Among subjects who arenot subject to the occupational swine exposure, it was further shownthat those residing downstream of swine farming district sustainhigher risk (792/1295, 61.2%) than those residing upstream (OR =1.29, 95%CI = 1.02∼1.64). It was concluded that swine are a princi-pal source of infection by genotype IV HEV in eastern China, humaninfection by the virus is acquired by contact with swine and the viruscould be disseminated via waterways to neighboring communities andthrough trafficking in pigs to more distant locations.

#100

Hbv DNA loads in different fibrosis stages, samevolume index of liver parenchyma and HCC innatural history of chronic hepatitis BWEI-MIN KE,1 LI-NA YU,1 SHI-BIN XIE,1

XIAO-HE LI,1 JING LAI,1 ZHI-LIANG GAO1

1Depatment of Infectious Diseases,The Third Affiliated Hospital,Sun Yat-Sen University

Aims The purposes are to clarify the status of HBVe system statusand HBV DNA loads in different fibrosis stages, same volume indexof hepatic parenchyma and HCC.Methods These studies detected and compared HBV e systemstatus and HBV DNA loads in different liver fibrosis stages, samevolume index of hepatic parenchyma and HCC.Results There were differences of age, HBeAg, anti-HBe positiverates and HBV DNA loads among liver fibrosis stages 1, 2, 3, 4 andHCC (P < 0.047∼0.000). There were not differences of HBV DNAloads apportioned by same volume index of hepatic parenchymaamong liver fibrosis stages 1, 2, 3 and 4 (P > 0.088∼0.953).Conclusions These results showed no change in HBV DNA loadsreleased by same volume index of hepatic parenchyma cells, it willhelp to institute the strategies and dosage of antiviral therapy againstchronic infection of HBV.

#101

Elevated serum tumor necrosis factor alpha andferritin may contribute to the insulin resistance foundin HCV positive Egyptian patientsMOHAMED ELSAMMAK,1,1 WAEL RAFAE,2

AMEL ELSAWAF,3 IBTESAM ABD-ELFATAH,4

EHAB ABD EL-ATTI,5 ABEER GHAZAL6

1Medical research institute, Department of Chemical Pathology,Alexandria university Egypt, 2Medical research institute,Department of internal Medicine, Alexandria university Egypt,3Medical research institute, Department of Chemical Physiology,Alexandria university Egypt, 4Medical reAlexandria universitystudent hospital, Department of internal medicine, Egypt,5Department of internal medicine, Menophya University Egypt,6Medical research institute, Department of Microbiology,Alexandria university Egypt

There is evidence of an increased incidence of insulin resistance anddiabetes mellitus (DM) in patients with hepatitis C virus (HCV)infection. Several mechanisms have been proposed, including inade-quate insulin secretion or interference with signaling within theinsulin receptor. We assessed serum tumor necrosis factor alpha(TNFalpha) and ferritin levels as potential mediators of insulin resis-tance in HCV positive Egyptian patients. PATIENTS ANDResults Patients (n = 27) with HCV infection, patients (n = 23)with hepatitis C and DM (HCV + DM), patients (n = 22) with DM,and sex- and age-matched controls (n = 18) were included in thisstudy. The degree of insulin resistance (HOMA index) was signifi-cantly higher in the HCV, HCV + DM and DM groups compared tothe controls. Serum TNFalpha levels were significantly higher in theHCV, HCV + DM groups compared with the healthy controls andDM patients (p < 0.001). There was a significant positive correlationbetween the HCV load and both HOMA index and TNF alpha level.HCV and HCV + DM patients also had significantly higher serumferritin levels compared with healthy controls and patients with DM.Conclusion Patients with HCV infection had a significantly higherlevel of TNFalpha and ferritin, which may explain their insulin resis-tance. HOMA index and serum TNFalpha levels correlated positivelywith the HCV load.

#102

Safety of two years therapy with adefovir dipivoxil(ADV) for Chinese patients with HBeAg positivechronic hepatitis B (CHB)MIN-DE ZENG,1 YI-MIN MAO,1 WEI-LUN LU,2

YU-MING WANG,3 YAO-ZONG WANG,4

JONATHAN S DIXON,5 KEITH F BARKER5

1Renji Hospital, Shanghai, PRC, 2Sun Yet-San 3rd Hospital,Guangzhou, PRC, 3Xinan Hospital, Chongqing, PRC,4Infectious Disease Hospital, Jinan, PRC, 5GlaxoSmithKlineR&D, Brentford, UK

Methods 480 Chinese HBeAg positive CHB patients (serum HBVDNA ≥ 106 copies/mL and ALT > 1 × ULN) were randomised 1 :2 :1 to receive placebo for 12 weeks then ADV 10mg for 40 weeksor ADV for 52 weeks or ADV for 40 weeks then placebo for 12 weeks(AAP group). 474 patients entered the 2nd year of study when allreceived ADV. Safety measures included adverse events (AE), seriousAEs (SAE), and laboratory assessments.Results 83% were males with a median age of 30yr. The mostcommon AEs were fatigue and rash. Study drug related AEs in 2 yearswere infrequent (5–12%) and mild or moderate in severity with lessincidence in 2nd year (0–3% in 2nd yr vs 5–11% 1st yr) in each of the3 groups. 14 patients prematurely discontinued with 6 cases related


to AEs. Only one drug related SAE (spontaneous abortion) wasreported. One death occurred due to gastric cancer.Over 2 years, there was no overall median change from baseline ineither serum creatinine or serum phosphorous in any group. Therewere no confirmed cases of serum phosphorous decrease to ≤1.4mg/dL.Three patients had confirmed >0.5mg/dL increase from base-line in serum creatinine, but none had a peak value out of normalrange.Most grade 3 and grade 4 ALT flares occurred in the AAP groupbetween weeks 40–52 when these patients were re-randomised toplacebo.Conclusion ADV for 2 years was safe and well tolerated in Chinesepatients with HBeAg positive CHB.

#103

Lack of impact of YMDD mutants on efficacy orsafety of two years therapy with adefovir dipivoxil(ADV) in Chinese HBeAg positive chronic hepatitis B(CHB) patientsYI-MIN MAO,1 MIN-DE ZENG,1 XIA-QIU ZHOU,2

HAO WANG,3 HONG REN,4 JUN-QI NIU,5

JONATHAN S DIXON,6 KEITH F BARKER6

1Renji Hospital, Shanghai, PRC, 2Ruijin Hospital, Shanghai,PRC, 3Beijing People’s Hospital, Beijing, PRC, 4CMU 2ndHospital, Chongqing, PRC, 5Jilin University 1st Hospital,Changchun, PRC, 6GlaxoSmithKline R&D, Brentford, UK

Methods 480 HBeAg positive CHB patients with serum HBVDNA ≥ 106 copies/mL and ALT > 1 × ULN were randomised toreceive placebo for 12 weeks followed by ADV for 40 weeks (PAAgroup, n = 120), or ADV for 52 weeks (AAA group, n = 240), or ADVfor 40 weeks followed by placebo for 12 weeks (AAP group, n = 120).474 patients entered the 2nd year of study and all received open labelADV. Prior treatment with lamivudine was permitted if therapy hadstopped ≥3 months before the study but some patients had YMDDmutant HBV (YMDDm) at baseline while all others had wild typeHBV (WT).This analysis investigated the impact of YMDDm at base-line on response to ADV. The analysis was done on a mITT popula-tion including all randomized patients with HBeAg positive CHB.Results The presence of YMDDm at baseline had little impact onweek 104 median log10 reduction of HBV DNA (PAA YMDDm −4.8[n = 24], WT −5.3 [n = 96]; AAA YMDDm −4.4 [n = 52], WT −5.2[n = 188]; AAP YMDDm −5.9 [n = 18], WT −4.5 [n = 102]), pro-portion of patients with ALT improvement (PAA YMDDm 95%,WT71%; AAA YMDDm 82%,WT 77%; AAP YMDDm 88%,WT 68%)or serological responses. There was also no impact on the incidenceor type of adverse events, laboratory toxicities, serum creatinine,phosphorus or other safety evaluations.Conclusion In Chinese patients with HBeAg positive CHB, base-line YMDDm status has no impact on the safety and efficacy of 2years ADV therapy.

#104

Overlap lamivudine treatment in patients withchronic hepatitis B receiving adefovir for lamivudine-resistant viral mutantsCHUN-JEN LIU,1 JIA-HORNG KAO,2 PEI-JER CHEN,2

MING-YANG LAI,2 DING-SHINN CHEN2

1National Taiwan University Hospital, 2National TaiwanUniversity College of Medicine

Adefovir dipivoxil is effective against lamivudine-resistant hepatitis Bvirus (HBV). Whether short-term overlap lamivudine is beneficialremains unknown, particularly in patients with decompensated

chronic hepatitis B. We enrolled 30 patients who received 48-weekadefovir (10mg daily) for hepatitis B exacerbation associated withlamivudine-resistant mutants. Nineteen (63.3%) patients had base-line evidence of hepatic decompensation. Lamivudine was combinedfor <1 months in 8 (group I), 2–5 months in 10 (group II) and >6months in 12 (group III).We analyzed their serial ALT levels, Child-Pugh (CP) score, serum viral load and lamivudine-resistant strains.We found that serum ALT became normalized in 20 (66.7%) andHBV DNA decreased to <100 copies/mL in 8 (26.7%) at the end of48-week treatment. The log(10) reduction of serum HBV DNA wassignificantly smaller in group I compared to group II and III patientsat week 24 and 48 of treatment [median (range): 3.0 (1.5–5.6) vs. 4.5(1.5–7.4), P = 0.032; and 3.4 (0.9–4.7) vs. 5.2 (2.2–7.7), P = 0.008;respectively]. In contrast, the virologic responses at the end of 48-week therapy were similar between group II and III patients. Theimprovement in serum ALT and CP score at week 48 was similar irre-spective of baseline decompensation, liver cirrhosis and the durationof overlap lamivudine therapy. Our findings suggested that an overlapof lamivudine for >2 months might achieve better virological but notbiochemical outcomes in patients receiving adefovir for lamivudine-resistance HBV. Since our case number was small and the study wasnot randomly controlled, further studies are needed.

#105

Is HCV genotype 3 associated with non-insulindepended diabetes mellitus?NIR HILZENRAT,1,2 KAHLID ALSAYARI,1,2

IAN SCHRIER,2,3 MARC DESCHENES,2,4

DONALD MURPHY,5 TINA KADER2,6

1Jewish General Hospital, GI Division, 2McGill University,3Jewish General Hospital, Department of Epidemiology, 4LiverDiseases Unit, Division of Gastroenterology, MUHC, 5Institutenational de sante publique du Quebec, 6Jewish General Hospital,Division of Endocrinology

Over the past few years, several studies showed an increased incidenceof Type 2 Diabetes Mellitus (T2DM) in patients with Hepatitis C(HCV). Up to 40% of HCV patients were found to have T2DM, asopposed to 2% in HBV patients.Non-Alcoholic Steatohepatitis has been recognized recently to be achronic liver disease that affects a substantial proportion of theworld’s population.This seems to be due, in part, to conditions asso-ciated with insulin-resistance state such as T2DM, obesity, and dys-lipidemia.Recently, it was shown that HCV genotype 3 is associated significantlywith steatohepatitis, more so than other genotypes of HCV. There-fore, we examined whether there is linkage between HCV genotype3 and T2DM, both of which are associated with steatohepatitis.Our cohort included 248 subjects with HCV. Thirty-six of thesepatients (15%) were found to have T2DM. The BMI of each patientwas calculated, and a pathologist assessed their liver biopsy fibrosisstage. Ten of the T2DM subjects and 46 of the patients withoutT2DM were found to have HCV genotype 3 (28% vs. 21% respec-tively, P = 0.42). However, multiple regression analysis revealed thathaving genotype 3 was associated with T2DM [OR (95% CI): 3.6(1.3, 9.8)] after adjusting for age, sex and BMI, but not the stage offibrosis [1.1 (0.5, 2.7)]. In conclusion: our cohort analysis suggeststhat having HCV genotype 3 is associated more with T2DM thanother HCV genotypes.


#106

The relationship between HBV genotypes and anti-Viral therapeutic efficacy of interferon-αLU-WEN WANG,1 ZUO-JIONG GONG1

1Department of Infectious Diseases, Renmin Hospital of WuhanUniversity

Aim To study the influences of HBV genotypes on anti-viral ther-apeutic efficacy of interferon-α in chronic hepatitis B patients.Methods The chronic hepatitis B patients, who were treated withα-IFN, were selected by expectation. The anti-viral therapeutic effi-cacies of these patients were observed. The HBV genotypes weredetected by PCR microplate hybridization ELISA. The levels ofserum HBV DNA were determined by fluorescent quantitative PCR.The HBV gene variation al sites of pre-C and BCP regions wereassayed by using technology of gene chip.Results The genotypes B and C were predominant in 94 chronichepatitis B patients selected by expectation. The genotypes A, E andF were not found in these patients. The HBV DNA levels in geno-type C and mixed genotype were significantly higher than in geno-type B. The response to α-IFN in patients of genotype B wereobviously excellent than genotype C and D, and complete responseto α-IFN was only observed in genotype B. The response to IFN-αin mixed genotype was the most insensitive compared with othergenotypes. The negative transition of HBeAg was correlated with thevariations of HBV pre-C and BCP regions in the patients of part-response or non-response to α-IFN. The variation rates of HBV pre-C and BCP region in genotype C were obviously higher than ingenotype B.Conclusion The regular detection of HBV genotypes in clinic willbe benefit for identification of disease prognosis and for choice of anti-viral therapeutic strategies.

#107

Epigallocatechin-3-gallate ameliorates alcohol-induced liver injury in ratsGUNGJIN YUAN,1 ZUO-JIONG GONG1


Aim To investigate the effect of epigallocatechin-3-gallate (EGCG)on alcohol-induced liver injury in rats, and to explore its mechanisms.Methods Ethanol and ethanol/EGCG group rats received fish oil(0.5ml) along with ethanol daily via gastrogavage for 6 weeks, whiledextrose and dextrose/EGCG group rats were given fish oil along withisocaloric dextrose instead of ethanol. The dextrose/EGCG andethanol/EGCG groups received additional treatment of EGCG (100mg/kg) daily intragastrically by gavage. ALT activity, TNF-αlevels and plasma endotoxin concentrations were assayed. TNF-α,CD14, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2(COX-2) mRNAs in the liver were analyzed by RT-PCR.Results Rats given fish oil plus alcohol developed fatty liver, necro-sis and mild inflammation, and an elevation in serum ALT activity.Treatment with EGCG resulted in absence of necrosis, slight inflam-mation in the liver and a decrease in serum ALT activity, but thedegree of fatty liver was unchanged.The amelioration of liver damagein the ethanol/EGCG group was accompanied by a reduction inserum TNF-α levels, plasma endotoxin concentratins, lipid peroxida-tion and TNF-α, CD14, iNOS and COX-2 mRNA expressions in theliver.Conclusion EGCG partially ameliorates alcohol-induced liverinjury. The mechanisms may involve the reduction in endotoxemia,lipid peroxidation, and CD14,TNF-α, COX-2 and iNOS expressionsin the liver.

#108

Effects of s-adenosylmethionine on liver methioninemetabolism and steatosis in rats with ethanol-inducedliver injuryPIN ZHANG,1 YAN-QING HUANG,1,1

ZUO-JIONG GONG1


Objective To investigate the effects of S-adenosylmethionine(SAM) on ethanol-induced liver injury in rats.Methods 48 female SD rats were randomly divided into 4 groupsas control, model, low dose and high dose SAM groups. Exceptcontrol group, all rats were fed high fat-containing diet plus ethanoland fish oil gavage for 8 weeks. SAM were administered by intraperi-tioneal injection after 4 weeks exposure of ethanol. Plasma totalhomocysteine(tHcy), serum aminotransferase activity (ALT), totalcholesterol (TC), triglyceride (TG), TNF-α, TGF-β levels, and livermalondialdehyde (MDA), and glutathione (GSH) contents wereassayed, Liver histology was also examined.Results Compared with control group, model group rats developedmarkedly liver damage, accompanied by an increase of tHcy, ALT,TC,TG,TNF-α,TGF-β and MDA levels, and decrease of GSH levels(P < 0.01). In treatment groups, SAM significantly protected the liverinjury, reduced ALT, TC, TG, MDA, TNF-α and TGF-β levels (P <0.05), and enhanced GSH levels (P < 0.01), but Plasma total homo-cysteine levels was not affected significantly (P > 0.05).There was nostatistical difference in the above parameters between the two dosegroups (P > 0.05).Conclusion SAM prevents alcohol-induced liver injury in rats byreducing liver lipid peroxidation, anti-inflammation and anti-hyperplasia. SAM does not affect the plasma total homocysteinelevels.

#109

Impact of Hepatitis B virus co-infection on responseof HIV-1 infected patients to highly activeantiretroviral therapy at the Jos University TeachingHospital(JUTH), NigeriaLADEP GWAMZHI,1,2 OCHE AGBAJI,1,2

PARTRICIA AGABA,1,2 PAM BADUNG,1,2

GODWIN IMADE,1,2 JEAN-LOUIS SANKALE,3

JOHN IDOKO,1,2 PHILLIS KANKI3

1Jos University Teaching Hospital, 2AIDS Prevention Initiativein Nigeria, 3Harvard School of Public Health, Boston, MA

Background Whether hepatitis B co-infection affects the responseof HIV-1 infected patients to therapy is not certain. We demonstratethe impact of hepatitis B on CD4 cell count recovery and HIV viralload reduction HIV-1 patients following commencement of anti-retroviral therapy.Methods This was a prospective study carried out between Dec2004 and Jun 2005 at JUTH. Three hundred and ninety seven con-senting HIV-1 patients completed the study. Their CD4 cell countsand HIV viral loads were done by Flow Cytometry and RocheAmplicor® respectively at three visits while receiving antiretroviraltherapy. The data obtained were subjected to analyses using Epi Info2004 software (Student’s t test and the Mann-Whitney test).Result Sixty-two of the patients (16%) were reactive to HBsAg.The mean age of HBV co-infected patients was comparable to thatof non-HBV infected patients. The mean CD4 cell recovery at 12weeks of therapy of HBV co-infected patients was 132 cells/microlitrewhile it was 98 cells/microlitre for non-HBV infected patients (p =0.05). Even though the median viral load reduction was higher fornon-HBV infected patients, this was not significant.


Conclusion An early rapid CD4 cell recovery among HBV co-infected patients was not sustained with a much more gradual HIVviral load reduction. HBV co-infection may thus have a negativeimpact on the response of co-infected patients to antiretrovirals.

#110

Autoimmune thyroid disease in patients with chronichepatitis B (HB) and hepatitis C (HC) before andafter interferon-alpha (IFN) therapy.BOGDAN MAREK,1 DARIUSZ KAJDANIUK,1

EWA JANCZEWSKA-KAZEK,2 BEATA KOS-KUDLA,3

LUCYNA SIEMIN*P1SKA,1 MARIUSZ NOWAK,1

HALINA BORGIEL-MAREK,4 ZOFIA OSTROWSKA5

1Dept. of Pathophysiology, Silesian Medical University, Zabrze,2Dept. of Infectious Diseases, Silesian Medical University,ChorzÖw, 3Dept. of Clinical Endocrinology, Silesian MedicalUniversity, Zabrze, 4Dept. of Maxillofacial Surgery, SilesianMedical University, Katowice, 5Dept. of Clinical Biochemistry,Silesian Medical University, Zabrze, Poland

The chronic hepatitis and IFN therapy display interactions with theimmune system. Thyroid dysfunction has been reported in patientswith viral hepatitis and during IFN therapy.Aim to describe the frequency and clinical outcome of thyroiddisease in HB and HC patients before and after IFN therapy. 40patients with HB, 40 with HC and 30 healthy subjects (C) werestudied. Usg examination of thyroid and assays of serum TSH, fT4,TPOAb were performed before and 6 months after treatment. Theoverall prevalence of thyroid disturbances was higher in patientsbefore therapy than in controls (3.3% in C vs 5% in HB vs 10% inHC). TPOAb titers was higher in HC compared to HB patients andC (38.7% in HC vs 24.5% in HB vs 10.4% in C). Increase serumTSH levels and hypoechogenic pattern of thyroid were more fre-quently in HC patients than in C (36 vs 6%). IFN therapy induceshypothyroidism (7.5% in HB vs 20% in HC).TSH levels were higherafter therapy in groups with HB (1.74 vs 3.06 μIU/ml) and HC (2.08vs 5.89 μIU/ml). At the end of IFN therapy TPOAb titers increasedin patients with HC (38.7 vs 69.4%) but not in those with HB (24.5vs 29.8%). HC can lead to latent autoimmune thyroiditis. Distur-bances of pituitary-thyroid axis were more frequent in HC than inHB patients. IFN treatment leads to hypothyroidism in some patientswith HC. Patients with HC treated IFN need systematic pituitary-thyroidal axis function testing.

#111

Predicting response to peginterferon alfa-2a (40KD)(PEGASYS®) in Asian patients with HBeAg-positivechronic hepatitis BGRAHAM COOKSLEY,1 WAN CHENG CHOW,2

TEERHA PIRATVISUTH,3 GEORGE K.K. LAU,4

PHILIP MCCLOUD5

1Clinical Research Department, Royal Brisbane Hospital,Brisbane, Australia, 2Gastroenterology Department, SingaporeGeneral Hospital, Singapore, 3Songklanagarind Hospital, Princeof Songkla University, Hat Yai,Thailand, 4Department ofMedicine, Queen Mary Hospital, University of Hong Kong,Hong Kong, China, 5Roche, Dee Why, Australia

Background and Aims Baseline ALT and HBV DNA levels aresignificant predictors of HBeAg response to peginterferon alfa-2a(40KD) (PEGASYS). The aim of this analysis was to develop andvalidate a simple practical tool to predict the probability of sustainedHBeAg response to peginterferon alfa-2a based on baseline HBVDNA and ALT levels.

Methods Logistic regression analysis was used to predict rates ofsustained HBeAg seroconversion 24 weeks post-treatment in 233Asian patients infected with genotypes B or C who were randomizedto treatment for 48 weeks with peginterferon alfa-2a 180 μg once-weekly in a large multicenter study. Predicted rates of sustainedHBeAg response were compared with observed rates.Results and Conclusion Predicted rates of HBeAg response con-curred well with observed rates (Table 1).This provides a simple wayto identify those patients with HBeAg-positive disease most likely tobenefit from peginterferon alfa-2a treatment in clinical practice inAsia.

Table 1 Predicted and observed rates of HBeAg response 24 weeks post-treatment

Predicted rates %[80% CI] Observed rates %

(n/n)

ALT ALT ALTHIGH MED LOW

Log10 HBV DNA 50% 38% 31%≤10.0 log [40–61] [31–45] [24–39]

52% 36% 32%(13/25) (22/61) (18/56)

Log10 HBV DNA 29% 20% 16%>10.0 log [21–39] [14–27] [10–23]

28% 22% 12%(8/29) (10/45) (2/17)

#112

Alanine aminotransferase (ALT) as an alternative toHBV DNA as a marker of sustained response topeginterferon alfa-2a (40KD) (PEGASYS®) treatmentTEERHA PIRATVISUTH,1 GEORGE K.K. LAU,2

ZHI-MENG LU,3 PATRIZIA FARCI,4 RUI JIN,5

CIHAN YURDAYDIN,6 JIAN WU,7 MATEI POPESCU8

1Songklanagarind Hospital, Prince of Songkla University, HatYai,Thailand, 2Department of Medicine, Queen Mary Hospital,University of Hong Kong, Hong Kong, China, 3Department ofInfectious Diseases, Ruijin Hospital, Shanghai, China,4Dipartimento di Scienze Mediche, Universita di Cagliari,Cagliari, Italy, 5Beijing You An Hospital, Beijing, China,6Faculty of Medicine, University of Ankara, Ankara,Turkey,7Roche, Dee Why, Australia, 8Roche, Basel, Switzerland

Background Persistently normal ALT or low serum HBV DNAhas been associated with a reduced risk of disease progression (Papatheodoridis et al. J Hepatol 2001; Chen et al. Hepatology 2005).Measurement of HBV DNA can, however, be costly. We investigatedthe correlation between ALT response and HBV DNA in patientsreceiving peginterferon alfa-2a (PEGASYS) to determine whetherALT can be used as a more cost-effective marker of sustainedresponse.Methods HBeAg-negative patients (n = 177) received PEGASYS180 μg once-weekly for 48 weeks. ALT and HBV DNA (Roche CobasAmplicor) were measured at 4, 24 and 48 weeks post-treatment.Results Patients with normal ALT 24 weeks post-treatment hadlower HBV DNA (n = 105; mean/median: 3.9/3.7 log10 cp/ml) thanthose without ALT normalisation (n = 72; mean/median: 6.3/6.6 log10 cp/ml). Patients with normal ALT were significantly morelikely to have HBV DNA <100,000cp/ml than those who had abnor-mal ALT (84% versus 26%, p < 0.001). In patients who had normalALT at 4, 24 and 48 weeks post-treatment (n = 36), mean/median


HBV DNA 48 weeks post-treatment was 3.9/4.0 log10 cp/ml in con-trast to 6.2/7.1 log10 cp/ml in patients who had elevated ALT (n = 14)throughout. HBV DNA was <100,000cp/ml in 81% of patients withpersistently normal ALT compared with 29% of patients with persis-tently elevated ALT (p < 0.01).Conclusion There was a strong correlation between ALT responseand low HBV DNA. ALT may therefore be an interesting alternativeto HBV DNA as a marker of sustained response, especially inresource-limited settings.

#113

Response-guided versus uninterrupted lamivudinetreatment to delay lamivudine resistance and need foradefovir in patients with chronic hepatitis B (CHB)SALVATORE MADONIA,1 FABIO TINÈ,2

GIUSEPPE MALIZIA,2 GANDOLFO GIANNUOLI,2

MIRKO OLIVO,3 MARIA RITA BADALAMENTI,1

GIUSEPPE CIVITAVECCHIA,1 GABRIELLA FASINO,4

DONATELLA FERRARO5

1Department of Internal Medicine AO “V Cervello”, Palermo,Italy, 2Department of Gastroenterology AO “V Cervello”,Palermo, Italy, 3Postgraduate school of GastroenterologyUniversity of Palermo, Palermo, Italy, 4Department ofMicrobiology AO “V Cervello”, Palermo, Italy, 5Istituto di IgieneUniversity of Palermo, Palermo, Italy

At our institution, before adefovir availability, patients with CHB andstable lamivudine on-treatment response (PCR-undetectable HBV-DNA, normal ALT), discontinued therapy at week 52 to avoid risksof lamivudine-induced mutations. Lamivudine was restored at relapse(HBV-DNA ≥1 × 105 copies). Since adefovir availability, lamivudineis continued until resistance (HBV-DNA detectable by PCR andYMDD mutation) and then adefovir is added. Unfortunately, ade-fovir is expensive and the risk of long-term mutation is not negligi-ble, making a delayed start desirable.To evaluate if the first approachcould enable a delay in lamivudine resistance, we compared a groupof consecutive patients treated with lamivudine in the pre-adefovirperiod according to the above-mentioned schedule (10 patients,group 1), with a subsequent group of consecutive patients, treatedwith lamivudine until resistance (19 patients, group 2).The groups 1and 2 were comparable according to age (45 and 48 years), sex (males70% and 84%), anti-HBe positive (90% and 84%) and cirrhosis(52.6% and 50%). Median follow up was 58 and 25mo respectively.Lamivudine resistance occurred in 5/10pts (50%) in group 1 and7/19pts (36.8%) in group 2. The median (range) time from firstlamivudine administration to YMDD was 29(18–50) mo and18(13–41) mo respectively. At relapse no patient experienced liverdecompensation. In patients with CHB with stable response tolamivudine, treatment withdrawal at week 52 with resumption atrelapse could sligthly delay resistance and need for adefovir. Thepotential benefit of this approach should by compared with the risksof decompensation at relapse.

#114

Specific genotype & autoantibodies patterns areassociated with higher degrees of inflammation &fibrosis in treatment naïve hepatitis C patientsATHINA CHOUNTA,1 CHRISTOS DRAKOULIS,2

SOTIRIOS TSIODRAS,1 CHRISTOS ELLINAS,1

SPYROS ZOURIDAKIS,1 ANTONIA ELEZOGLOU,1

KOSTAS PONTIKIS,1 FANI PLIARCHOPOULOU,1

ELENI PAPA,3 MARGARITA THEODOROU,4

HELEN GIAMARELLOU5

1Hepatology Clinic, Attikon General Hospital, University ofAthens Medical School, 2Hepatology Clinic, General Hospital ofPireaus, 3Immunology Laboratory, General Hospital of Pireaus,4Molecular Biology Laboratory, General Hospital of West Attica,54th Department of Medicine, Attikon General Hospital,University of Athens Medical School

Background Histological correlates of autoantibody detectionaccording to genotyping & viral load have not been widely exploredin treatment naïve hepatitis C patients.Methods All naïve pts from 2 large liver centers underwent liverbiopsy & testing for antinuclear (ANA), anti-smooth muscle (SMA),anti-mitochondrial (AMA) & anti-liver-kidney-microsomal (LKM1)antibodies.Viral load was quantified using RT-PCR & genotyping wasperformed by RT-PCR & hybridization. Liver biopsy was graded &staged according to Ishak. Correlations between autoantibody detec-tion, genotyping, viral load & histological findings were examined byunivariate & multivariate analysis.Results 124pts (54.8% male) were studied. ANA, SMA & AMAwere detected in 33.9%, 21.8% & 1.6% of pts respectively. LKM werenot present. Genotype-1 was detected in 46.8% pts & a viral load > 800,000 copies/ml in 55.6% pts. (+) ANA were more common ingenotype-1 vs. other genotypes (p = 0.008). pts with (+) ANA & (+)SMA had higher inflammatory grades (p < 0.01). Greater inflamma-tion grade was associated with increasing age (OR: 1.04, 95% CI1.01–1.06), (+) SMA (OR: 3.3, 95% CI: 1.2–8.4), and viral load > 800,000 copies/ml (OR: 2.3, 95% CI: 1.01–5.1) while genotype-1approached significance (OR: 2.1, 95% CI: 0.95–4.7).Conclusions Increasing age & genotype-1 were associated withANA detection in this cohort. Greater inflammatory changes wereassociated with age, SMA presence & higher viral load. Further elu-cidation of correlations between virus characteristics, autoantibodypresence & their role in hepatitis C related histological damage is necessary.

#115

Atopy and the Yin/Yang of Th1/Th2 influence on HBVmutation and eliminationMICHAL R. PIJAK,1 FRANTISEK GAZDIK,1

STEFAN HRUSOVSKY,12 MARIAN OLTMAN2

1Slovak Medical University, Bratislava, Slovakia, 2Departmentof Gastroenterology, University Hospital, Bratislava, Slovakia

Background Similarly to atopy, HBeAg-positive chronic hepatitisB (CHB) is also characterised by a skewing of the immune systemtowards a Th2 phenotype. Thus, we aimed to determine whether thepresence of atopy might influence HBeAg/antiHBe status and theoutcome of antiviral therapy.Methods We studied 73 treatment-naive patients with CHB, 39men and 34 women aged 16–60 years (mean 32 years) with a posi-tive HBV-DNA assay (Digene). Serological markers HBeAg and anti-HBe were tested by ELISA (MONOLISA, Sanofi Pasteur). Theatopic status was determined on the basis of 2 or more positive skinprick tests from a panel consisting of 7 aeroallergens. After baselineevaluation, all patients were treated with interferon-alpha (IFN) 10MIU thrice weekly for 24 and 48wks respectively, depending onHBeAg status.Results The prevalence of atopy in the entire cohort was 34%. Inthe subgroup of HBeAg-positive patients, the prevalence of atopy wassignificantly higher than in the HBeAg-negative patients (61.5% vs.2.9%, p < 0.0001, Chi-square test). One year after the completion ofthe treatment, HBV-DNA was undetectable in 32.8% of all patients.The proportion of responders was significantly higher among atopicpatients than in non-atopic patients (52% vs. 22.9%, p = 0.012, Chi-square test).


Conclusions Consistently with the Th1/Th2 paradigm, our resultsdemonstrated an inverse association between anti-HBe positivity andatopy. This finding and a higher response rate to IFN in atopicpatients lead us to conclude that both strong Th1 (Yin) and syn-chronous co-stimulatory Th2 (Yang) activities may be required for asuccessful HBV elimination.

#116

Linked precore/core mutations associated withexacerbation of hepatitis B: a dynamic study on HBV quasispeciesLIN LIU,1 LIN LAN,1 JUN-GANG LI,1

GUO-HONG DENG,1 JIE XIA,1 JI-JUN ZHOU,1

XIAO-HONG WANG,1 YU-MING WANG1

1Institute of Infectious Diseases, Southwest Hospital

Background/Aims The clinical significance of mutations in thecore promoter (CP) and precore (preC) regions of hepatitis B virus(HBV) genome remains controversial.The relationship between thesemutations and those in the core (C) region has rarely been studied.Methods The dynamic changes of composition of HBV quasi-species were analyzed in serial serum samples from a patient with twoepisodes of exacerbation and resolution of chronic hepatitis B, utiliz-ing a method of PCR—TA cloning—conformation sensitive gel elec-trophoresis (CSGE)—sequence analysis.Results Five of the total seventeen samples (three during exacer-bation, two during resolution) were extensively studied. Thirty-threeHBV clones from each sample were CSGE screened to identify clono-types. Different clonotypes in each sample were sequenced. The dataprovided evidence of crossing-over between previous dominant andminor sequences under different host immune pressures. A significantlinkage was revealed between four mutations, core P5T, L60V, S155Tand precore G1896A mutations (P < 0.001). The data also indicateda significant correlation of these mutations with the exacerbation ofhepatitis B (P < 0.001).The CP A1762T/G1764A joint mutation wasassociated with neither the other four mutations nor exacerbation ofhepatitis.Conclusions (1) Composition of HBV quasispecies changes underdifferent environmental pressures. (2) There is a linkage between coreP5T, L60V, S155T and precore G1896A mutations. (3) These muta-tions are significantly associated with exacerbation of hepatitis B.

#117

The influence of HBV genotype on the efficacy oflong–term lamivudine therapy in patients with HBeAgpositive chronic hepatitis BMEI ZHU1

1Jing an qu central hospital, Shanghai, China

Background and Aims Recent studies have indicated thatpatients with HBV genotype B is associated with better response tointerferon therapy and good prognosis in HBeAg(+) chronic hepati-tis B than genotype C. However, the influence of HBV genotypes onrate of HBeAg/anti-HBe seroconversion and durability after long-term lamivudine administration was inconclusion. This study was toevaluate the relationship between HBV genotype and the factorswhich may affect the efficacy of lamivudine therapy.Methods A total of 167 HBeAg and HBV DNA positive CHBpatients took lamivudine 100mg once daily more than 3 years, thenfollowed up for more than 1 years after cessation of the treatment.The baseline serum were genotyped by PCR used six pares of type-specific primers in HBV S gene and results were confirmed by DNAsequencing. SASS software was adopted in data analysis.Results The distribution of HBV genotype among 167 patientswere genotype B, 38 (22.75%); genotype C, 124 (74.25%); mix geno-

type (B + C), 2 (1.2%) and undetection, 3 (1.8%), but there waswithout statistical significance (p > 0.05). During more than 3 yearsof lamivudine therapy, the HBeAg seroconversion rate of genotype Band genotype C were (31.6% vs. 25%), there was no statistical sig-nificance (p = 0.802, p > 0.05). The seroconversion in patients witheither B or C were durable (83.2% vs. 84.0%, p > 0.05).Conclusions This study did not revealed obvious influence ofHBV genotype on the efficacy of long-term lamivudine therapy inHBeAg positive CHB patients.

#118

Durability of seroconversion after long-termLamivudine therapy in HBeAg positive chronichepatitis B patientsXIUYUN MA1

1The outpatient department of Beijing ditan hospital, Beijing,china

Objective The aim of this study is to monitor the factors on thatmay impact the rate of HBeAg seroconversion and its durability afterlong-term lamivudine therapy in patients with HBeAg positivechronic hepatitis B.Methods 167 patients with HBeAg positive CHB were treatedlamivudine 100mg daily for more than 3 years. Once HBeAg sero-conversion occurred more than two times (once every 3 months), thepatients continued dosing for 6–12 months, then stopped lamivudineand were followed-up for more than 1 year. At every month of year1, and every 3 months thereafter, HBV serologic markers, HBV DNAlevel, alanine aminotransferase (ALT), and YMDD mutation weretested. HBV genotyping was performed by type-specific PCR. Thedata were analyzed by Logistic multivariant analysis.Results Among 167 patients 45 cases achieved HBeAg serocon-version (27.0%). The annual accumulative seroconversion rates were10.2%, 16.7%, 23.3%, 25.1% and 27.0% in 1st, 2nd, 3rd, 4th and5th years, respectively. After more than 1 year of followed-up moni-toring, 9 patients developed reactivation with reappearance of HBeAgand elevation of HBV DNA. The 1-year durability of HBeAg sero-conversion after discontinuing Lamivudine therapy was 80.0%(36cases). Logistic multivariate analysis showed that the rate of HBeAgseroconversion and its durability correlated with a high baseline ALT.In contrast, a relatedly low seroconversion and durability rate wasobserved in patients with high baseline HBV DNA and YMDD mutation.Conclusion Continuation of lamivudine therapy for more than 6months after HBeAg seroconversion might increase the durability ofresponse.

#119

Estimating utility of hepatitis B premarital screening:target population attitudePEYMAN ADIBI,1 SAEED HEDAYATI,1

GHOLAMHOSSSEIN SADRI,1

ELHAM FAGHIH-IMANI1

Spouse to spouse transmission of hepatitis B via sexual andhorizontal routes, which is present in Iran with a relative risk ofabout 3, may be prevented by active and passive immunizationof at-risk spouse.

During a formal premarital health screening and education program,all men and women attended in two separate rooms and after a pre-sentation about hepatitis B (Including epidemiology, transmissionand preventive methods, health impact and complications) a validatedgroup-administered questionnaire was distributed.


The sample population was 1316 people (50% woman) with the ageaverage 24.39 ± 4.63yrs. Within the sample 72.3% said that they areready to be screened in their questionnaire but when they asked toput their names on the paper and actually be screened only 18%remained. Ninety percent preferred to know their fiancé potentialpositive result and let their fiancée know theirs. On a situation of pos-sible test result, 31.8% still intended to marry and 29% still wantedto marry with their fiancée if his/her test would become positive.Thirty percent of cases expressed they will be depressed if theirfiancée would not marry them due to positive hepatitis B result.Although hepatitis B premarital screening was found to be cost-effective in Iran, but this study showed that it may face major socialresistances and may cause significant personal impacts. Although theresponders theoretically preferred screening but their real practice didnot toward it in more than one-fifth of them. The idea of “not tomarry if the test is positive” in about 70% of instances is a matter ofconcern in implementation of a regular screening program in Iran thatmay guide us to modification of prevention method.

#120

Experience with Acute Liver Failure from New Delhi:etiology, outcome and prognostic indicators—comparison with international referencesSHASHIDEEP SINGHAL,1 ASHOK SEHGAL,2

SHASHI SHARMA,2 PREMASHIS KAR1

1Department of Medicine, Maulana Azad Medical College andLN Hospital. New Delhi., 2Institute for Cytology and PreventiveOncology (ICMR), Noida, U.P.

The course of Acute Liver Failure (ALF) is usually unpredictable andassociated with a high mortality. Etiologies and outcome differ geo-graphically, thus there is need to identify prognostic markers tailoredfor particular regions. 203 consecutive patients 82 males and 121females (pregnant 67) with ALF during 1994–2005 from a tertiarycare hospital were analyzed for etiology, clinical and biochemicalparameters which were compared in survivors and non-survivors ina univariate analysis followed by multivariate analysis to determinethe prognostic markers.Viral infections were the commonest cause ofALF, hepatitis E, B, A and C contributing 52%, 27%, 8% and 2%respectively. Mixed viral etiologies 7% and anti-tubercular drugs 3%were also implicated, whereas in 14% no specific etiology was found.Other hepato-tropic viruses like TTV, SEN-V and GBV-C werestudied in limited patients by PCR but had limited significance.Amongst 65% who died causes of mortality were; cerebral edema51%, coagulopathy 21%, infections 18% and renal failure 9%. Basedon results of univariate analysis, multiple logistic regression analysiswas employed to find the independent risk factors of which PT-difference >20, liver-span <3cms, age >40 and ALT > 1000IU/L weresignificantly (p < 0.01) associated with mortality in that order of sig-nificance. Comparison of data with other Asian, European and American countries revealed PT and age as common prognosticmarkers in all geographic regions of world. Optimization of prognos-tic markers in specific populations would help in better patient selec-tion and timely referral to a transplant centre.

#121

Seroprevalence of HBV in children and adults forestimating of national vaccine program, Malekan city,Iran 2004SEYED HASSAN MONTAZAM,1

ASGAR TANOMAND,2 AMIR AFSHIN KHAKY,2

ALI ASGAR SANY,2 MEHDI NAJARY NABI2

1Bonab islamic azad university, Iran, 2Tabriz university ofmedical sciences

Purpose of studyHepatitis B is the important factor of liver cancer and has significantmortality worldwide.The risk of acquiring HBV infection is 1–15% in the most areas.A large number of cases are seen in Middle East.MethodsIn this study, blood samples of 200 (100 children and 100 adults)were collected.First of all, HBsAg were estimated by EIA.Then, the people who hadpositive samples, were examined for HBeAg, HBeAb, and IgM anti-HBc.The results of children and adults compared with each other bySPSS version 11.ResultsThe total number of adults who had positive results was 5 cases (5%).Nobody of them was not vaccinated. Then we did HBeAg, HBeAb,and IgM anti-HBc on the sera of HBsAg positive individuals.HBeAb was positive in 4 (80%) cases. IgM anti-HBc and HBeAgwere negative in all cases.All of children had negative results.ConclusionsAccording to the results of this research, the prevalence rate of HBVcarriers in studied cases (adults) were 5%. This result is well corre-sponded with the previous studies in same areas.Due to the IgM anti-HBc was negative in all HBsAg positive cases,it was demonstrated that, individuals did not have current infectionwith HBV. In the mention of HBeAb were positive in 80% of carri-ers, it was showed that these persons had past infection with HBV,Vaccination induced 0% prevalence among children that is similar todeveloped countries.It is recommended vaccination for children and high risk groups andeducational programs about HBV.

#122

Increasing trend of acute hepatitis A in north India:Need for identification of high-risk population for vaccinationPREMASHIS KAR,1 ZAHID HUSSAIN,2

SYED A HUSAIN,3 NANDAGUDI S MURTHY,4

BHUDEV C DAS4

1Department of Medicine, Maulana Azad Medical College andLN Hospital. New Delhi, 2PCR Hepatitis Laboratory,Department of Medicine, Maulana Azad Medical College.New Delhi., 3Department of Biosciences, Jamia Millia Islamia.New Delhi., 4Institute of Cytology and Preventive Oncology,Noida, U.P.

Background Hepatitis A (HAV) is endemic in India and most ofthe population is infected asymptomatically in early childhood withlifelong immunity. Because of altered epidemiology, decreasingendemicity, the pattern of acute HAV infection is changing in India.Aim of the study was to assess whether the proportion of adults withacute HAV infection has been increasing over the years and to analyzeseroprevalence of IgGanti-HAV antibodies in adults above the age of15 years who were otherwise asymptomatic in order to determinewhether the seropositivity is changing in adult population.Methods Sera of 3495 patients of acute (1932) and chronic (1563)liver disease at LN-Hospital during 1999–2003 were tested for sero-logical markers of acute (HBsAg, HBcIgM, anti-HCV, HEV-IgM,and HAV-IgM) and chronic (HBsAg, HBcIgG, HBeAg, and anti-HCV) hepatitis. 500 normal healthy attendants above the age of 15years were tested for IgG anti-HAV as controls.Results Of 1932 patients with Acute Viral Hepatitis, 221 (11.4%)were positive for IgM Anti-HAV.The patients who were IgManti-HAVnegative included hepatitis B (321) patients, C (39 Patients), E (507patients) and unclassified (844 patients).The frequency of HAV infec-tion had increased from 10.6% to 22.0% among children and from


3.4% to 12.3% among adults in 5 years period. 98% (294/300)patients with chronic liver disease were positive for IgGanti-HAV antibody.Conclusions Although universal vaccination against HAV is notcurrently indicated, selective vaccination of the high-risk population,based on their serological evidence of HAV antibody, would be a ratio-nal and -cost effective approach.

#123

The influence of HCV NS5A on α-IFN signal pathwayGUOZHONG GONG,1 JIE CAO2

1Second Xiangya Hospital, Central South University, 2SecondXiangya Hospital Central South University

Objective To explore the influence of HCV NS5A protein on theSTAT-1 phosphoration and nuclear translocation induced by IFN-αand understand the molecular mechanism of HCV resistant to IFNtherapy. Methods Hepatocellular carcinoma cell line Huh7 was tran-siently transfected with HCV NS5A protein expression plasmidpCNS5A for 48h and then interacts with IFN-α2b (5000 IU/mL) for30m. Indirect immunoflouresent staining was employed to detectSTAT-1 phosphoration and nuclear translocation, as control, unin-fected and empty plasmid pRc/CMV infected Huh7 cells wereincluded.Results Within uninfected and pRc/CMV infected, after IFN-α2bstimulation for 30m, the almost all Huh7 cells were STAT-1 phos-phorated and nuclear translocated, but, within pCNS5A transfectedHuh7 cells, STAT-1 phosphoration and nuclear translocation weregreatly reduced.Conclusion IFN-α2b is able to activate STAT-1 phosphorationand nuclear translocation and this effect is inhibited by HCV NS5Aprotein, indicating HCV NS5A interferes with IFN-α signal pathway,which may be the involved in HCV resistant to IFN therapy.

#124

Hepatitis C Virus NS5A Protein Upregulates SurvivinGene ExpressionGUOZHONG GONG,1 YANG ZHOU1

The Center for Liver Diseases,The Second Xiangya Hospital,Central South University, Changsha 410011, China

Objective To investigate whether the nonstructural 5A protein(NS5A) encoded by the human hepatitis C virus RNA genome affectsthe expression of survivin gene.Methods HCV NS5A expression plasmid (pCNS5A) was transfectedinto HepG2 cells with lipofectin reagent and HCV NS5A protein wasconfirmed by immuno- histochemistry stainning. Survivin mRNAwas evaluated by reverse transcriptase polymerase chain reaction (RT-PCR) and the expression of survivin protein was detected by westernblot experiment. Results HCV NS5A protein was detected in thecytoplasm of the HepG2 cells transfected with pCNS5A, and the expres-sion of survivin mRNA and survivin protein was both up-regulatedin the presence of HCV NS5A protein.Conclusions HCV NS5A protein can stimulate survivin proteinexpression, and the possible way is by strengthening the survivin genetranscription.

#125

Relationship between the expression of HO-1 withoxidative stress in nonalcoholic steatohepatitisYU LIN SONG,1 HUIZHONG GAN,1 NAIZHONG HU,1

JIAN MING XU,1 MIN PAN1

1Department of Gastroenterology,The First Affiliated Hospital ofAnhui Medical University, Anhui, China

Objective To investigate the expression of heme oxygenase-1 (HO-1) in the rat liver of nonalcoholic steatohepatitis and the relationshipof HO-1 with oxidative stress.Methods 20 male Wistar rats were randomly divided into controlgroup which fed with standard diet and model group with high fatdiet. The pathologic changes of all rat liver was scored using lightmicroscopy in the end of 12WK. The aspartate aminotransferase(AST), alanine aminotransferase (ALT), triglyceride (TG), total cholesterol (TCH), malondialdehyde (MDA) and GSH-peroxidase(GSH-px) were measured by biochemical methods. The expressionof HO-1 was detected by immunohistochemical method.Results Compared with control group, the model group developedmiddle-heavy steatosis, serum ALT; AST,TG,TCH were significantlyincreased (P < 0.05, P < 0.01), TG, TCH, MDA in liver tissuehomogenates were also significantly increased (P < 0.01), and GSH-Px in liver tissue Homogenates were significantly decreased (P <0.05). HO-1 expression was increased in model group and correlatedwith the increase of MDA (r = 0.672, P = 0.033) and the decrease ofGSH-Px (r = −0.703, P = 0.023).Conclusions HO-1 expression was increased and probablyinduced by oxidative stress and lipid peroxidation in nonalcoholicsteatohepatitis.

#126

Effects of melatonin on oxidation, antioxidation innonalcoholic steatohepatitis rats fed with high fat dietHUIZHONG GAN1,YU LIN SONG1, NAI ZHONG HU1,JIAN MING XU1, MIN PAN1

1Department of Gastroenterology, the First Affiliated Hospital ofAnhui Medical University, Anhui, China

Objective To investigate the effects of meltatonin on hepatic mor-phology, lipid and the system of oxidation and antioxidation in non-alcoholic steatohepatitis rat.Methods 50 male Wistar rats were divided into 5 groups and 10per each randomly. The meltatonin in low does, moderate does, highdoes group and model group were fed with high fat diet, and controlgroup with common dietfor 12 weeks. Meltatonin (contain ≤1% dehy-drated alcohol) was intraperitoneally injected to The rats in variousmelatonin groups (2.5mg/kg, 5mg/kg and 10mg/kg, respectively) andequal does of saline (contain 1% dehydrated alcohol) to control andmodel group once a day. Histopathological changes in liver wereobserved. The level of serum aspartate aminotransferase (AST),alanine aminotransferase (ALT), liver tissue Homogenates triglyc-eride (TG), total cholesterol (TCH), superoxide dismutase (SOD),malondialdehyde (MDA) and GSH-peroxidase (GSH-px) were mea-sured by biochemical methods.Results Compared with control group, the model group developedmiddle-heavy steatosis; ALT, AST,TCH and MDA contents were sig-nificantly increased (P < 0.05), SOD and GSH-px contents were sig-nificantly decreased (P < 0.05), Compared with model group, ALT,AST, TCH contents decreased and the pathological changes in liverwas improved in melatonin-treated groups; the decrease of MDA (P< 0.001) and the increase of GSH-Px, SOD (P < 0.001, 0.02 < P <0.05) was correlated with the increase of MT does.


Conclusions MT significantly improved morphology, amelioratedantioxidase activity and lipid peroxidation of fatty liver, which demon-strated the protective and therapeutic effect for nonalcoholic steatohepatitis.

#127

Impact of HCV, HBV infections in renal chronicpatients from hemodialysis centers in RomaniaELENA LAURA ILIESCU,1,1

MIHAELA IAVORENCIUC,2 MIHAI VOICULESCU2,1

1EASL, 2Fundeni Institute

Introduction Several studies have shown that HCV and HBVinfections have an important impact in the haemodialysis patients.Epidemiological reports from our country are contradictoryAim We studied the distribution, evolution of HCV, HBV infec-tions in haemodialysis patients. This study include a large number ofpatients, with a broad geographical distribution.Materials and Methods Is the first study which include viralquantification and genotype determination for HBV infection. The177 patients were tested for anti-HCV, HBsAg, anti-HBc, anti-HBe,HBe Ag by MEIA Axsym, ABBOTT.Viral load was measured by PCRHBV monitor assay and PCR HCV monitor assay Serum HBV DNAwas expressed as copies-ml with the lower limit of detection <200copies/ml For HBV genotyping Line Probe Assay was used.The lowerlimit of detection of HCV RNA was <600UI/ml.Results The HCV prevalence was 28.24%, HBV prevalence was5.08% and HBV + HCV 1.12%. We found genotype D in thehaemodialysis patients.We observed that a higher number of patientshad normal ALT levels: 61,01% from anti-HCV positives patients,77,78% from HBs Ag positives. We treated 15 patients with Peg-Interferon alpha-2a (Pegasys) 135 μgSQ/week. The virologicalresponse was 75%.Conclusion Patients at haemodialysis constitute a high-risk groupfor HCV, HBV infectionThe antiviral treatment is associated with ahigher rate of virological response.

#128

Association of interleukin-10 gene polymorphisms andoutcome of hepatitis B virus infection, the first reportfrom IranHOUSHANG RAFATPANAH,1 REZA MIRZAEI,1

HAMIDREZA SIMA,2 NARGES VALIZADEH,1

MOHAMMAD MOEINI,2

ABOULFAZL MAHMOODZADEH,2

KAMRAN GHAFARZADEGAN,2

MOHAMMAD KHAJEDALOUEE,3

ABBAS ESMAILZADEH,2 ABBAS BOSKABADI,2

KARIM JALAEIAN,2 JALIL TAVAKKOL AFSHARI1

1Bu-Ali Research Institute, Mashhad University of MedicalSciences, Mashhad, Iran, 2Gastroenterology Research Center,Mashhad University of Medical Sciences, Mashhad, Iran,3Department of Community Medicine, Mashhad University ofMedical Sciences, Mashhad, Iran

The role of immunologic factors in Hepatitis B virus (HBV) infec-tion is not fully understood. In a case-control study 81 patients withchronic HBV infection (26 inactive carriers, 26 chronic hepatitis, and29 cirrhotic patients) and 177 healthy volunteers were enrolled.Threebiallelic polymorphisms (-1082/-819/-592) in the IL-10 gene pro-moter were analyzed by amplification refractory mutation system

(ARMS)-PCR method. There was no statistically significant differ-ence in the genetic polymorphisms between three divided patientgroups and the healthy control group (Table 1). Although we founda decrease in the frequency of A/A and an increase in frequency ofG/G genotypes at position -1082 in three groups of cirrhotic patients,chronic hepatitis, and inactive carriers, respectively, it was not statis-tically significant (Figure 1).It was concluded that Polymorphisms of IL-10 gene (-1082/-819/-592) do not play any role in the outcome of hepatitis B infectedIranian patients.

Table 1 Genotype distribution of IL-10 gene promoter polymorphisms

Loci Genotype Cirrhotic Chronic Inactive Healthy P valuePatients hepatitis carriers controln(%), n(%), n(%), n(%),n = 29 n = 26 n = 26 n = 177

−1082 AA 13 (44.8) 11 (42.3) 7 (26.9) 71 (40.1) 0.752AG 14 (48.3) 11 (42.3) 15 (57.7) 91 (51.4)GG 2 (6.9) 4 (15.3) 4 (15.4) 15 (8.5)

−819 CC 13 (44.8) 8 (30.7) 13 (50) 95 (53.7) 0.127CT 11 (37.9) 14 (53.8) 13 (50) 69 (39)TT 5 (17.2) 4 (15.3) 0 (0) 13 (7.3)

−592 CC 13 (44.8) 8 (30.7) 13 (50) 95 (53.7) 0.127CA 11 (37.9) 14 (53.8) 13 (50) 69 (39)AA 5 (17.2) 4 (15.3) 0 (0) 13 (7.3)

44.848.3

6.9

42.3 42.3

15.3

26.9

57.7

15.4

40.1

51.4

8.5

0

10

20

30

40

50

60

Cirrhoticpatients

Chronichepatitis

Inactivecarriers

Healthycontrol

AA

AG

GG

Per

cent

age

Groups

Figure 1 Differential distribution of IL-10 (−1082) genotypes.

#129

Increase CD4+/CD25+ T cells frequency and functionoccur after HBV vaccine administration at protective titerALESSANDRO PERRELLA,1,2 LUIGI RACIOPPI,3

SIMONA PISANTI,3 LAURA VITIELLO,3

SALVATORE DE SIMONE,3 COSTANZA SBREGLIA,2

PASQUALE BELLOPEDE,2 TOMMASO PATARINO,2

LUGI ATRIPALDI,2 ORESTE PERRELLA2

1Dpt Infectious Disease and Immunology, 2Dpt Infectious Diseaseand Immunology, Hospital D.Cotugno, 3Dpt Biologia e PatologiaCellulare e Molecolare, Federico II Medical School university

Background/Aim The immunoregulatory mechanisms of sero-conversion occurring in patients undergoing HBV vaccination are not


well known. Aim of the present study was to assay frequency andfunction of CD3+/CD4+/CD25+ T cells after HBV vaccination at theprotective titer of HBs-Ab.Methods We enrolled 10 subjects undergoing three doses ofHBsAg vaccine (Engerix-B DNA-recombinant) injected intramuscu-larly in the deltoid region at time 0 (T0), one (T1) and sixth month(T6), following the manufacturer’s instructions. CD3+/CD4+/CD25+lymphocytes were enumerated in PBMC using flow cytometry(Beckman Coulter), according to previous evidences (MS Longhi JHepatol 2004;41:31–7). T lymphocytes activity was assessed usingimmunomagnetical sorting with magnetic bids (purified Tregs >95%)(Dynal Treg kit) as capacity to inhibit the proliferation ofCD4+/CD25− T cells stimulated with anti-CD3/CD28 (1 :2 and 1 :20 ratio).Results Subjects were administered vaccine after one monthachieved a protective titer of Anti-HBs (>100IU/mL) and had a con-temporary statistically significant increase of CD4+/CD25+ frequency(T0 = 47 ± 11 cells; 7% ± 1% vs T1 177 ± 53 cells; 25% ± 8% UMann Whitney Test p < 0.05) and function (Ratio 1 :2 = 84% vs 98%;Ratio 1 :20 = 12% vs 25%)Conclusion An increased CD3+/CD4+/CD25+ T cells frequencyand function seems to occur after one month of vaccine administra-tion when a HBsAb protective titer is achieved, suggesting a role ofCD4+/CD25+ T regulatory cells in the seroconversion and propos-ing them as a possible tool to monitor antiviral treatment response.

#130

Elevated CD4+/CD25+ regulatory T cell frequency andfunction in acute hepatitis C genotype 1, presagechronic evolutionALESSANDRO PERRELLA,1 LUIGI RACIOPPI,2

LUIGI ATRIPALDI,1 LAURA VITIELLO,2

SIMONA PISANTI,2 PASQUALE BELLOPEDE,1

TOMMASO PATARINO,1 RAFFAELE VELA,1

TIZIANA ASCIONE,1 ORESTE PERRELLA1

1Dpt Infectious Disease and Immunology, Hospital D. Cotugno,2Dpt Biologia e Patologia Cllulare e Molecolare, Federico IIMedical School University

We decided to assess CD4+/CD25+ T cells frequency and function,in all patients admitted in our regional Infectious disease center. Weperformed our assay according to previous evidences on Tregs (MSLonghi J Hepatol 2004;41:31–7) by means of flow citometry assayassaying their function at standard proliferation assay after immuno-magnetically sorting with magnetic bids (Dynal kit).Results We admitted 12 patients with acute hepatitis C (genotype1; Group A) following for six months their parameters and compar-ing them to 10 healthy individuals (Group B). 10/12 patients in groupA had a significant elevation of Treg frequency (124 ± 38 cells; 13%± 3%) and function (ratio 1 :2 = 100%; 1 :20 = 99%) compared togroup B (6% ± 1.5 %; 42 ± 16; ratio 1 :2 = 80%; 1 :20 = 70%) (UMann-Whitney test z = 2.56; p < 0.001; z = 3.12; p = 0.001; z = 2.32;p < 0.01).Those patients did not show any decrease in their Treg fre-quency and function after six months, having also positivity for HCV-RNA and hypertransaminasis.The remaining two patients from groupA (patient 1: 8%; 96 Treg cells; patient 2: 7%; 87 Treg cells) did notshow any difference with healthy donors at the admission having negative HCV-RNA with normal transaminases after six months.Conclusion Increased Tregs number and function at the time ofacute hepatitis in the HCV patients who developed chronic hepatitissuggests that these cells may predispose to chronic evolution.

#131

Frequency of the different genotypes of hepatitis Cvirus among Libyan patients attending two tertiarycare hospitals in LibyaABDUL-NASSER ELZOUKI,1 ABDUNABI ALRYES,2

BASHIR ALAGI,2 MOHAMAD ALARABI1

1Faculty of Medicine, Garyounis University, Benghazi, Libya,2Faculty of Medicine, Alfateh University,Tripoli, Libya

Objectives To determine the distribution of HCV-genotypesamong patients attending two tertiary care hospitals in Benghazi andTripoli, Libya, and to correlate this with viral load.Methods One hundred and forty three consecutive HCV-RNApositive Libyan patients were recruited for the study. HCV genotyp-ing was carried out using the line probe assay (LiPA). Clinical detailswere elicited from patients’ hospital records.Results HCV genotypes 4, 1, 2, 3, and mixed infections (withgenotypes 1 and 4) were found in 63.6%, 19.6%, 11.9%, 3.5%, and1.4% of our patients, respectively. Genotypes 5 and 6 were notdetected. HCV genotype distribution was homogeneous, except forgenotype 4 that was detected with greater frequency in patients fromEast Libya as compared with patients from West Libya (76.3% Vs41.6%, p = 0.001). Similarly, genotype 1 was detected more fre-quently in patients from West Libya as compared with its detectionin patients from East Libya (33.3% Vs. 12.9%, p = 0.001). Parenteraltransmission accounted for 56% of all infections. There was no sig-nificant correlation between HCV genotype distribution and the viralload.Conclusions Genotype 4 was the most prevalent HCV genotypein Libya, especially in the Eastern part. No correlation between HCVgenotype distribution and the viral load was found.

#132

Prevalence of hepatitis B and C infection in Libya: A population-based nationwide seroepidemiological studyABDUL-NASSER ELZOUKI,1,2 MOHAMAD SMEO,3,2

MOHAMAD SAMOOD,3,2 ALSADEK ABONAJA,3,2

BASHIR ALAGI,4,2 MOHAMAD DAW,5,2

ABDULRAHMAN FORRARA,5,2 ICHRIS AHMAD,2,6

ABDULHAFID ABODHAIR2,7

1Department of Medicine, Faculty of Medicine, GaryounisUniversity, Benghazi, Libya, 2CDC, Libya, 3Department ofCommunity Medicine, Faculty of Medicine, Alfateh University,Tripoli, Libya, 4Department of Medicine, Faculty of Medicine,Alfateh University,Tripoli, Libya, 5Department of Microbiology,Faculty of Medicine, Alfateh University,Tripoli, Libya,6Department of Anatomy, Faculty of Medicine, AlfatehUniversity,Tripoli, Libya, 7Department of Gynacology,Faculty of Medicine, Alfateh University,Tripoli, Libya

Aims The present study was aimed to establish the prevalence ofcirculating hepatitis B surface antigen (HBsAg) and anti-hepatitis Cvirus (Anti-HCV) antibodies in a population-based survey in LibyanJamahiriya.Methods This study was part of the National Serum Survey(2004–2005) that conducted by the National Center for InfectiousDisease Control and Prevention, and included a random selection of65.711 individuals of different age groups by using multistage sam-pling technique. All subjects completed a face-to-face interview togather data on demographics and self-reported risk behaviours. Serafrom all participants were tested for HBsAg and Anti-HCV antibodiesusing third-generation enzyme-linked immunosorbent assay (ELISA)techniques.


Results The seroprevalence rates of HBsAg and Anti-HCV anti-bodies were 2.18% and 1.19%, respectively.Conclusion The results suggest the need of continues implement-ing of stringent measures for prevention and control of these infec-tions in Libya, including screening focused on high risk groups ofpopulation, precise determination of risk factors for transmission ofthese infections, and continuous of HBV vaccine to all newborns andhigh risk groups.

#133

Antiviral therapy and clinical outcome ofcompensated HCV cirrhosis with and without portal hypertensionVITO DI MARCO,1 PIERO LUIGI ALMASIO,1

STEFANIA DE LISI,1 VINCENZA CALVARUSO,1

DONATELLA FERRARO,1 DONATELLA FERRARO,2

SERGIO PERALTA,1 PIETRO PARISI,1

GIUSEPPE ALAIMO,1 NICOLA ALESSI,1

ROSA DI STEFANO,2 ANTONIO CRAXÌ1

1Unit of Gastroenterology and Hepatology, University ofPalermo, Italy, 2Department of Virology, University of Palermo,Italy

Background and Aim We evaluated the benefits of antiviraltherapy on clinical course of HCV cirrhosis with and without portalhypertension. Patients and methods. 174 consecutive patients withChild-Pugh A cirrhosis (mean age 57 years; 62% males; 56% withoesophageal varices; 88% genotype 1; 67% naïve to antiviral therapy;33% previously treated with IFN monotherapy.) were treated withPeg IFN (27%) or Peg IFN plus RBV (73%) and followed for amedian of 24 months (range 6–53). Results. Fifty-nine patients (34%)withdrew treatment because of side effects. Sustained virologicalresponse (SVR) was obtained in 18% patients by intention to treatanalysis. No significant difference among patients without and withvarices (21.1% vs. 15.5%, p = 0.3) was found. Logistic regression afterROC curve analysis confirmed as independent predictor of SVR theage ≤ 62 years (R.R. 5.6, 95% C.I. 1.1–29.5) the genotype 2 or 3(R.R. 19.1, 95% C.I. 5.3–67.8), the basal ALT > 3 ULN levels (R.R.3.9, 95% C.I. 1.4–11) and the basal GGT < 1 ULN (R.R. 4.6, 95%C.I. 1.6-12-7). During follow-up, 33 patients (19%) developed clin-ical events related to liver disease. Thirty-one events occurred in nonresponders and only 2 events in patients with SVR (21.7% vs 6.5%p < 0.05). Conclusion. PEG-IFN and Ribavirin can be efficacy evento patients with compensated cirrhosis and portal hypertension. SVRis more frequent in younger patients with genotypes 2 or 3 and ele-vated ALT. Patients with SVR have a minor incidence of disease com-plications during the follow-up.

#134

Expression of hepatitis B virus gene in patients withchronic hepatitis B from different nationalities inethnic minority areas in Yunnan, China in E.coliZHONG-QI BIAN,1,2 ZHAN-LOU HUA,1

WEI-YAO YAN,2 MING-QIU LIU,2

ZHAO-XIN ZHENG,2 DIAN-YUAN WU1

1Center for Infectious Diseases, Kunming General Hospital ofPeople Liberation Army, Kunming, 2State Key Laboratory ofGenetic Engineering, Institute of Genetics, Fudan University,Shanghai

Objective To investigate the function of HBV preS2/S and C genesof diverse-nationality patients with HBV in Yunnan.Methods HBV DNA were extracted from serum-samples of 50CHB patients (25 diverse-nationality patients with CHB and 25 Han

people patients of controls) from ethnic minority regions in Yunnan,China. HBV preS2/S and C genes were obtained by PCR. HBVpreS2/S and C genes were inserted into vector pBS and sequenced.HBV preS2/S and C genes from 50 cases were 846 and 552nucleotides (nt) in length. Compared with the HBV sequences in theGenBank database, HBV preS2/S and C genes among all the subjectswere homologous to ayw1 in sequence. Then HBV preS2/S and Cgenes were cloned into the expression vector pλPR, and recombinedpλPR-S2S and pλPR-C were constructed and transformed into E.coliTOP10 to express.The corresponding expressive non-fusion proteinswere identified through the methods of SDS-PAGE and Western blot.Results HBV genotype B was identified in all patients. High-levelexpression of E.coli TOP10 for two non-fusion proteins with relativemolecular weights of 31000 and 21000 were determined by SDS-PAGE.Conclusion HBV strains in patients with CHB from differentnationalities in ethnic minority areas in Yunnan, China was found tobe geneotype B (subtype ayw1). HBV genotype B were not relevantto different nationalities. The two non-fusion proteins encodedpreS2/S and C genes in the research proved to have antigenicity by Western blot. Findings of the study have for the first time revealed the S antigen amino acids (aa) 121∼147 withCRTCTTPAQGTSMFPSCCCTKPMDGNC epitopes, the Cantigen aa 75∼83 with NLEDPASRE epitopes, and the aa 107∼118with CLTFGRETVLEY epitopes, which were recognised by mono-clonal antibody IgG respectively among patients with CHB from dif-ferent nationalities in ethnic minority areas in Yunnan Province, P.R.China.

#135

Identification of hepatitis B virus genotypes inpatients with chronic hepatitis B from differentnationalities in ethnic minority areas in YunnanProvince, P.R. ChinaZHONG-QI BIAN,1,2 ZHAN-LOU HUA,1

WEI-YAO YAN,2 MING-QIU LIU,2 DIAN-YUAN WU,1

ZHAO-XIN ZHENG2

1Center for Infectious Diseases, Kunming General Hospital ofPeople Liberation Army, Kunming, 2State Key Laboratory ofGenetic Engineering, Institute of Genetics, Fudan University,Shanghai

Objective To determine whether there were evolutionary differ-ence of HBV genotypes and clinical significance among diverse-nationality CHB patients.Methods HBV preS2/S and C genes were inserted into vector pBSafter being amplified by PCR from HBV DNA, which were obtainedfrom serum-samples of 50 CHB patients (25 diverse-nationalitypatients and 25 Han people patients of controls) from ethnic minor-ity regions in Yunnan Province, P.R. China, the cloned preS2/S andC genes were sequenced.Results HBV preS2/S and C genes from 50 cases were 846 and552 nucleotides (nt) in length. Compared with the HBV sequencesin the GenBank database, HBV genotype B was identified in allpatients. HBV genotype B did not find statistically differencesbetween two groups (P > 0.05). In all cases, HBV S genes were foundto be Arginine (AGA) at nt 518–520, Lysine (AAA) at nt 632–634,and AGA and AAA were found at the corresponding amino acids (aa)122th and 160th, respectively. In all the subjects, aa variations of HBVC genes was found among codons 27∼63, 80∼110, and 135∼153involved in T and B cell epitopes.Conclusion It was the first time that HBV epidemic strains hadbeen identified as genotype B (subtype ayw1) in the ethnic minorityareas of Yunnan Province, P.R. China. HBV genotype B were not rel-evant to different nationalities. From the history of the ethnic minori-ties in Yunnan Province and the genetic analysis, HBV genotype B in


this area was for the first time identified as geographic original strain,and association of genotype B infections with the severity of liver dis-eases and curative effect. Besides, HBV viral load was found to be theonly significant variable associated with the CHB patients’ prognosis.

#136

Chinese marmota—a new animal model for HBV infectionDONGLIANG YANG,1 BAOJU WANG,1 XINYU LI,1

YONGJUN TIAN,1 MENGJI LU,2,3

MICHEAL ROGGENDORF2

1Division of Clinical Immunology,Tongji Hopsital of TongjiMedical College, HUST, China, 2Institut für Virologie,Universitätsklinkum Essen, Essen, Germany, 3Department ofMicrobiology,Tongji Medical College of HUST,Wuhan, China

478 of sera from Chinese marmots were collected from different loca-tions in China and tested by anti-WHc ELISA.Though some samplesshowed reactivity in anti-WHc ELISA, PCR amplification with con-served primers for hepadnavirus did not generate positive results.Hybridization by using a full length WHV as probe at a low strin-gency did not indicate the existence of WHV-like virus in collectedsamples.The susceptibility of two marmot species to WHV was tested. TwelveMarmota Himalaya and eight Marmota bobak were inoculated intra-venously with WHV. Sera samples were collected up to 40 weeks postinoculation. After inoculation with WHV, all 12 Marmota Himalayashow WHcAb positive (100%), 8 animals (75%) show WHsAg andWHV DNA positive. WHsAg and WHV DNA persisted in 7 animalsuntil death for at most 37 weeks, and became negative only in oneanimal at week 35 post inoculation. WHV DNA replication interme-diates, pregenomic RNA and WHsAg expression were detected inliver samples by Southern-blot, Northern-blot analysis and immuno-histochemical staining, respectively. All 8 Marmota bobak remainedWHcAb negative after inoculation with WHV.Conclusion 1) Our date do not support the early reports about theexistence of WHV-like virus in Chinese marmots. 2) WHV is able toinfect one Chinese marmot species despite the narrow host range ofhepadnaviruses.

#137

Packaging RNA (pRNA) as an escort for siRNA tointerfer HBV replicationDONGLIANG YANG,1 ZHONGJI MENG,1

ZHENGMAO ZHANG,1 YONGJUN TIAN,1 YAN YANG,1

PEIXUAN GUO2

1Division of Clinical Immunology,Tongji Hospital of TongjiMedical College, HUST, China, 2Laboratory of Gene Therapy,Purdue University, HANSEN B-036, USA

Small interfering RNA (siRNA) has been demonstrated considerablepotential as therapeutic agents for the treatment of hepatitis B virus(HBV) infection. However, the lack of a suitable targeted deliverysystem has seriously hampered any possible therapeutic applications.The DNA-packaging nanomotor of bacterial virus phi29 contains sixcopies of ATP-binding pRNA molecules.This pRNA is a unique andversatile molecule that can help prevent two problems commonlyassociated with therapeutic RNA molecules: exonuclese degradationand misfolding in the intercellular environment. The objective of ourcurrent study is to explore the potential use of pRNA as an escort forsiRNA to inhibit HBV gene expression and replication. We havedesigned a series of pRNAs carring small interfering RNA (siRNA)targeted to various regions of HBV genome and first tested the effi-cacy of these siRNA in suppression of HBV gene expression and repli-

cation both in vitro and in vivo. siRNA directed to the coding regionof HBsAg (siHBsAg) were very effective in knocking down the expres-sion of co-transfected HBsAg. Furthermore, 6–8-week-old BALB/cmice were used, and 15ug pHBV1.3 plus 1nmol pRNA-siHBV wereinjected into the tail vein by hydrodynamic injection. HBV antigensexpression and replication in the animal liver were significantly inhib-ited by intruducing the pRNA-siHBV molecule. Our preliminary datashowed that pRNA was an ideal vector for the delivery of siRNA tointerfere HBV expression and replication.

#138

A substitution at the amino acid position 122 ofHBsAg strongly impairs it’s antigenicityYONGJUN TIAN,1 ZHENHUA ZHANG,1 YANG XU,2

ZHONGJI MENG,1 MENGJI LU,2,3

DONGLIANG YANG1

1Division of Clinical Immunology,Tongji Hospital of TongjiMedical College, HUST,Wuhan, China, 2Department ofMicrobiology,Tongji Medical College of HUST,Wuhan, China,3Institute of Virology, University Hospital of Essen, Essen,Germany

Mutant HBsAg (mtHBsAg) with amino acid (aa) substitutions withinthe a-determinant like G145R are known to affect the binding of spe-cific anti-HBs antibodies and their detection by conventional diag-nostic assays. Recently, new types of mtHBsAg with aa substitutionsR/K122I or R/K160N were reported. These aa positions play animportant role for determination of HBV serotypes, thus particularlyrelevant for anti-HBs binding. In the present study, the binding ofanti-HBs to 16 different mtHBsAg with aa substitutions at 23 differ-ent sites within HBsAg a-determinant including G145R and R/K122Iwere tested. The reactivity of 14 monoclonal anti-HBs antibodies(MAb1 to MAb14) to mtHBsAg was measured by enzyme-linkedimmunoassay. 14 MAbs presented different patterns of detection ofmtHBsAg. MAb1 (D12F10E2) and MAb2 (1C2) reacted stronglywith 13 of 16mtHBsAg. Particularly, these 2 MAbs showed high reac-tivity to mtHBsAg with G145R that was not detected by a series ofcommercial anti-HBs assays. However, none of the 14 MAbs presentbinding affinity with the mtHBsAg with R/K122I in the culture super-natant or the cell lysate. Immunofluorescence (IF) staining of cellsexpressing mtHBsAg with R/K122I revealed that this mtHBsAg wasrecognized by polyclonal anti-HBs but not by MAbs. In conclusion,mtHBsAg with G145R showed reduced antigenicity but was recog-nized by some newly generated MAbs. MtHBsAg with R/K122I sub-stitution could not be detected by any MAbs, indicating that theantigenicity of mtHBsAg was strongly impaired.

#139

Study on immunogenicity of hepatitis B vaccine by 0-7-14-day scheduleSHI ZHU CHEN1

1Department of Infectious diseases, Chinese PLA 451 Hospital,Xi’an

Objective To raise seroconversion rate quicker.Methods 732 normal subjects were randomised into two groups.Group 1 370 cases aged 5∼68 years old. 20ug (10ug for ≤10 years)HB vaccine was vaccinated intradeltoideusly, as 0-7-14-day schedule.Group 2 362 cases aged 5∼69 years old. HB vaccine were vaccinatedwith 0-1-6-mon. schedule with same dosage as group 1. The condi-tions were comparable between two groups. All of subjects were testedfor anti-HBs at 1, 3, 7, 12, 24, 36 months after first dose.Results Positivity rate of anti-HBs at 1, 3, 7, 12, 24, 36mon. afterfirst dose were 97.0, 97.3, 94 (p < 0.01 vs group 2), 3, 85.1, 66.2,51.4% in group 1, and 51.9, 68.3, 79.1, 77.8, 69.5, 55.7% in group


2, respectively. The geometric mean titer (GMT) at 1, 3, 7, 12, 24,36mon. were 112.7, 117.3 (p < 0.001 vs group 2), 82.4, 67.1, 41.6,18.2 IU/L in group 1, and 51.3, 69.5, 115.1, 80.7, 49.4, 24.8 IU/L ingroup 2, respectively. The seroconversion rate of anti-HBs were93.1% in group 1 and 12.7% in group 2 (p < 0.001), respectively, inthose who had not anti-HBs after previous 0-1-6-mon. schedule.66.7% in group 1 and 10.0% in group 2 who had infected HBV andhad not produced anti-HBs after loss of HBV marks anti-HBs becamepositive (p < 0.05), respectively.Conclusion The seroconversion rate and GMT was markedlyhigher in o-7-14-day schedule than in 0-1-6-mon schedule 3mon.after first dose. Repeat vaccination with 0-7-14-day schedule for non-responder after 0-1-6-mon schedule have a high seroconversion rate.The positivety rate of anti-HBs is higher with 0-7-14-day than with0-1-6-mon schedule for those who had not anti-HBs after HBV marksdisappearance.

#140

High prevalence of occult hepatitis B in patients withadvanced liver disease and patients with hepatitis CRAOUF AHMED ABBAS,1 EL SAEED HALA,1

NAGLAA GHNAIEM,1 ASHRAF BASSUNY,1

HOSAM EL-EZZAWY,1 IMAM WAKED1

1National Liver Institute, EGypt

Reports indicate the higher prevalence of occult hepatitis B amongpatients with chronic hepatitis C, and that occult HBV might influ-ence the severity of disease. This has not been studied among Egypt-ian patients with chronic HCV genotype 4.Aim to evaluate the presence of HBV-DNA in HBsAg negativepatients with chronic liver disease, and to correlate its presence withHCV infection, and with severity of liver disease.Patients and Methods 224 HBsAg negative patients with liverdisease and 227 individuals without liver disease had serum HBV-DNA tested using nested PCR by specific primers of surface antigen.Patients were: 185 HCV genotype 4 positive (86 with liver cirrhosis,58 with chronic hepatitis, 41 with persistently normal ALT) and 39HCV negative (14 with liver cirrhosis and 25 with chronic activehepatitis).Results HBV-DNA was positive among 53 of 100 patients withliver cirrhosis (54% in HCV+ve vs 43% in HCV-ve, ns. HBV wasmore prevalent among patients with liver disease than controls(OR45, 95% CI: 11.59–382), and among patients with advanced thanmild liver disease (cirrhosis vs CAH OR8.78, 95% CI 3.4–25.2).Conclusion Occult hepatitis B is highly prevalent among patientswith hepatitis C and with liver disease compared to control, and theprevalence increases with advanced liver disease. Among patients withsimilar degree of liver disease the presence of HBV-DNA is not asso-ciated with increased markers of disease severity. The role of occulthepatitis B in the progression of liver disease remains to be identifiedin future studies.

#141

Hepatitis B and C viral markers among familymembers of patients with advanced liver disease andoccult hepatitis BAHMED ABBAS RAOUF,1 HALA EL-SAEED,1

NAGLAA GHNAIEM,1 ASHRAF BASSUNY,1

HOSAM EL-EZZAWY,1 IMAM WAKED1

1National Liver Institute, Egypt

The risk of infectivity of HBV among relatives of patients with occultHBV remains unknown. The aim of this study was to examine theHCV and HBV serological markers and viral status of immediatefamily members of HBsAg −ve patients with liver cirrhosis.

Patients and Methods 100 HBsAg −ve cirrhotic patients provided4 consenting family members each. Patients and family memberswere tested for HBsAg, anti-HBs, anti-HBc, anti-HCV, HCV-RNA,and HBV-DNA using.Results 86 Patients were +ve for HCV, and 53 were HBV-DNA +ve(48 sero+ve and 5 sero-ve). Among the 400 relatives, 122 had anti-bodies to HCV (30.5%), 12.25% were HBsAg +ve, and 3.25% hadoccult hepatitis B. Patients with occult hepatitis B had 18.9% of theirrelatives HBV-DNA +ve vs 11.7% of the relatives of HBV-DNA −vepatients. Patients −ve for HBV and HCV markers had none of theirrelatives +ve for any HBV markers. HBV markers were not differentamong siblings, spouses and offsprings (occult HBV in 6.5%, 1.9%and 4% respectively, ns).Conclusion Family members of patients with HBsAg −ve chronicliver disease are at higher risk of being +ve for HCV than HBV. Thisrisk is higher if the patient is HCV +ve than occult hepatitis B +ve,and is highest if both are positive. Overt HBV was the main form ofHBV infection in immediate family of patients with occult HBV.Thissuggests that occult HBV is a later stage in the disease progress ofHBV infection following overt HBV.

#142

The comparison prevalence of HBs Ag in medicalstaffs with asymptomatic blood donorsMEHDI BABAEI1

1Semnan University of Medical Sciences

Introduction There are 350 million chronic carriers of HBV in theworld. The 3 percent of people are carrier state in Iran. The impor-tant complications of the disease are cirrhosis and Hepatocellular carcinoma.Material and Methods collecting the blood donors and medicalstaffs information’s, in a questionnaire.Results the prevalence HBs Ag between the 15507 donors has been1/3 percent in 1998 to 2000. This amount has been 1/35 percent in1998 and 0/9 percent in 1999 to 2000. This number of contamina-tions at bloody group and Rh is the from of (AB− > B+ > O+ > A− >B− > O− > AB+), the most high prevalence has seen in the joblesspeople and the students had the least prevalence of HBs Ag, HBs Agbetween 113 personnel of medical staffs has been 1/7 percent. 47percent of personnel have done vaccination and the most group arephysicians and the most few group of them are servant.The influenceHBV vaccination between the personnel is 71/6 percent, in the male73/9 percent and in female is 70 percent. 31 percent of personnel arecontact with HBV but only the 1/7 percent of them were antigencarrier.Discussion HBs Ag prevalence is 1/03 percent that is the leastpercent between the other cities of Semnan province. Semnan hassecond position in low prevalence area in the country.The prevalenceHBs Ag in hospital personnel is 1/7 percent, that it is meaningful morethan blood donors (p = 0.05).

#143

Predictive value of hepatitis B virus cccDNA inPBMC of response to lamivudine therapy in patientswith chronic hepatitis BHAO-YU GUO,1,2 DE-MING TAN,1 XU-WEN XU1

1Department of Infectious Diseases, Xiangya Hospital, CentralSouth University, Changsha, Hunan, China, 2Department ofHepatology, Hunan provincial people Hospital, Changsha,Hunan, China

Objective To assess the value of hepatitis B virus (HBV) covalentlyclosed circle DNA (cccDNA) in peripheral blood mononuclear cell


(PBMC) for the prediction of the sustained response to lamivudinetherapy in patients with chronic hepatitis BMethods Seventeen chronic hepatitis B patients with complete orpartial response (serum HBV DNA turned negative, and ALT wasnormalized with or without HBeAg/anti-HBe seroconversion) tolamivudine treatment were selected. HBV-cccDNA in PBMC wasdetected at the end of lamivudine treatment, and serum HBV-DNAwere followed up for 1 year after treatment at three months interval.Results Nine out of 17 patients were positive for HBV cccDNA intheir PBMC at the end of lamivudine treatment. Virologic relapsewere found during 1 year follow-up in all of these 9 patients; whereasonly 1 relapse was observed among the 8 patients without HBVcccDNA.Conclusion HBV cccDNA in PBMC at end of treatment is of goodpredictive value for the sustained response to lamivudine treatmentin chronic hepatitis B patients.

#144

Inhibition of the secretion of HBsAg and HBV DNA invitro by hepatitis B Immune globulinZHONG-TIAN PENG,1 DE-MING TAN,1

SHUN-LING HUANG,1 ZHOU-HUA HOU,1

FEI LIU,1 PING-AN ZHU,1 RONG-QUAN FU1

Objective To investigate Hepatitis B Immune Globulin (HBIG)endocytosis into hepatocytes and inhibition of the HBsAg and HBVDNA secretion in vitro.Methods HBIG in DMEM were added to the monolayer grouthof QSG7701 and HepG2 2, 2, 15 cells.The supernatant at some daysinterval after incubation with and without HBIG was collected forHBsAb, HBsAg quantitative detection by time-resolved immunoflu-orometric assay and HBV DNA quantitative assay by realtime-PCR.MTT was used to evaluate the cytotoxicity of HBIG.Results HBIG in the concentration from 0.1 IU/mL to 5.0 IU/mLhad no cytotoxicity to the cells of QSG7701 and HepG2 2, 2, 15.55% of HBIG was endocytosed into QSG7701 after 48 hours ofculture. HBsAg in supernatants of HepG2 2, 2, 15 with 0.1, 1.0 or5.0 IU of HBIG/ml was reduced by58, 71 or 78% and HBV DNAreduced by 19, 20 or 23%, respectively, compared to HepG2 2, 2, 15without HBIG after three days incubation. At 5th, 7th and 9th daysculture with 1.0 HBIG/ml, the amount of HBsAg secreted in super-natants was reduced by 77, 90 or 92%, and HBV DNA was reducedby 50, 58 or 65%, respectively, as compared to the control.Conclusion HBIG can be endocytosed into hepatocytes and caninhibit the secretion of HBsAg and HBV DNA in vitro.

#145

Incidence of acquiring hepatitis C virus infection inpatients on hemodialysisNAGLAA ALLAM,1 BASSELOS SAAD,1

AHMED ABBAS,1 IMAM WAKED,1

SALEH MAHMOOD SALEH1

1National Liver Institute

Background and Aim Viral hepatitis is a major hazard for patientsand medical personnel in hemodialysis centers. This study was con-ducted to study the incidence of acquiring hepatitis C viral (HCV)infections in hemodialysis patients to settle the question of whetherdialysis is a risk of viral hepatitis infection.Methods Ninety patients starting hemodialysis treatment were fol-lowed up for six months. None of the patients received blood trans-fusion. Each of these patients was subjected to analysis of anti-HCVAbs using third generation ELISA to detect HCV infection at the startof and six months after dialysis. Group I was subdivided into 2 groupsaccording to the preliminary serological studies. The patients were

distributed in 3 dialysis units, each having a peculiar protective strat-egy. Unit A isolates infected patients in a separate room and on ded-icated machines. Unit B devotes machines dedicated for HCV virusinfected patients. Unit C does not isolate HCV infected patients.Results Results revealed that the incidence of HCV was 23.3%.Units that isolate HCV infected patients or use dedicated machinesfor them had significantly lower rates. In unit A and B, the incidencewas 15.16% and 13.3% respectively versus 41.66% in unit C.In Conclusion De novo infection by HCV is relatively high amonghemodialysed patients and does not seem related to blood transfu-sion. Our results strongly suggest the need for strict preventioncontrol measures in addition to isolation of serologically positive HCVpatients, a policy that still needs further evaluation.

#146

Compared the effect of lamivudin in the hepatitis Bpatients between HBeAg-negative group and HBeAg-positive groupJIANNING JIANG,1 MINGHUA SU,1

YUANPING ZHOU,2 JIZHOU WU,1 ZHIHONG LIU,1

YING HUA WEI,1 LIYI HUANG,1 YANHONG YU1

1The Dept. of infectious disease, the first hospital of Guangximedical university, 2Hainan medical college

Aim Compared the curative effect, recurrence of HBV-DNA afterlamivudine therapy in chronic hepatitis B (CHB) with HBV-DNApositive between HBeAg-positive group and HBeAg-negative group.Methods Both patients of HBeAg-positive and HBeAg-negativereceived lamivudine (100mg daily) for more than 1 year with mea-suring liver function, HBV-DNA and HBsAg, HBsAb, HBeAg,HBeAb, HBcAb of serums before therapy, in every month aftertherapy until HBV-DNA transferring negative, then every threemonths, if discontinuation of lamivudine measuring in every monthfor six months, and then every 3 months. The study was conductedby retrospective and prospective analysis.Results The average therapy course was 22.66 moths in 59 patientsof HBeAg-positive and 22.45 months in 42 patients of HBeAg-negative (p > 0.05); The time of HBV-DNA transfering negative wassimilar in HBeAg-positive group and HBeAg-negative group, respec-tively 4.36 moths and 4.76 moths (p > 0.05);The rate of therapy end-point achieving (according to the China lamivudine treatmentguidelines) in HBeAg-positive group were lower than in HBeAg-negative group, respectively 30 patients (50.8%) and 35 (83.3%) (P = 0.01). The rate of HBV-DNA recurrence in HBeAg-positivegroup were higher than HBeAg-negative group, respectively 36patients (61.0%) and 15 patients (35.7%) (P = 0.012).Conclusion lamivudine therapy induces a similar primary virologyresponse in HBeAg-positive and HBeAg-negative CHB. On the otherhand, HBeAg-negative CHB is easy to achieve therapy end-point andhad lower rate of HBV-DNA recurrence than HBeAg-positive CHB.

#147

A research of the non-blood transfused hepatitis C infectionWEI-MIN SHE,1,2 DE-CHANG HU,1,2

CHAO-HUI ZHOU1,2

1Zhongshan Hospital, 2Fudan University

Objective To survey non-blood transfused hepatitis C infectionamong different groups in Shanghai.Method

1. Investigate 1645 persons, including 99 drug users, 411 medicalworkers, 826 ordinary persons, and 309 blood donors.

2. The serum was extracted from the blood samples to detect theHCVAb by EIA.


Results

1. The entire group: HCV infection among the drug users, medicalworkers, ordinary persons and blood donors was 38.4% (38/99),4.62% (13/411), 0.98% (8/826) and 0.96% (3/3.9) respectively.

2. The drug user group: HCV infection among intravenous injectiondrug users was 60.8% (31/51), which was obviously higher thanthe of drug users without intravenous injections (14.6%, 7/48).Among the intravenous injection drug users, the infectious rate ofthe drug users who shared needles was 86%, which was higherthan that of those did not share (50%).

3. The medical worker group: HCV infection among dentistryworkers was 10.4% (11/106), which was significantly higher thanother medical workers (2.62%, 8/305). Infectious rate of dentistryworkers from 4 hospitals was 3.3% (1/30), 0% (0/17), 14.6%(7/48) and 27.3% (3/11), respectively.

4. The ordinary person group: HCV infection among adults was1.57% (7/446), higher than that among children (0.27%, 1/370).

Conclusion The highest rate of non-blood transfused hepatitis Cinfection in Shanghai was found in the drug user group (38.4%), andthe second high was found in dentistry workers (10.4%). The infec-tious rate was found to be very low among ordinary persons and blooddonors (both <1%).

#148

Immune responses in mice induced by bicistronicDNA vaccinces containing hepatitis B virus coreantigen and Flt3 ligand geneYONGLIN YANG,1 JUN LI,1 QIBIN HE,1

YIPING XING,1 ZUHU HUANG1

1Jiangsu Province Hospital

Objective To construct bicistronic DNA vaccines encoding hepati-tis B virus core antigen (HBcAg) and extracellular fragment of Flt3ligand (FL) and observe its immunogenecity in mice.Methods HBcAg and FL genes were cloned into vector pJW4303at upstream (pJW4303/C/FL) and downstream (pJW4303/FL/C) ofinternal ribosome entry site (IRES) element, seperately. 293 T cellswere transfected with this DNA vaccines to examine the expressionsof HBcAg and FL. Five groups of BABL/c mice were vaccinated withDNA vaccines. Anti-HBc IgG titers and HBcAg specific Th1/Th2type cytokines secreted by spleno-lymphocytes were measured byELISA method and ELISPOT assay accordingly.Results All bicistronic DNA vaccines expressed both HBcAg andFL in vitro and expression level was higher when target gene locatedupstream of IRES element. The titers of anti-HBc IgG and levels ofTh1/Th2 type cytokines in mice immunized with pJW4303/C/FLwere significantly higher or stronger than that with pJW4303/C andpJW4303/FL/C.Conclusions Bicistronic DNA vaccines encoding HBcAg and FLwere constructed.The expression of target gene at upstream of IRESelement is definitely better than that at downstream. The immuno-genicity of HBcAg DNA vaccine is enhanced by the introduction ofFL gene.

#149

Effects of alcohol intake on patients with chronichepatitis BMOBIN WAN,1 SHANGYOU HONG,1

XUESONG LIANG1

1Department of infectious diseases, Shanghai Changhai Hospital

Objective To investigate roles of alcohol intake on hepatic injuriesin patients with chronic hepatitis B.

Methods 84 patients were screened from the data bank, anddivided into 3 groups of alcohol intake, simple chronic hepatitis B,chronic hepatitis B accompanied with alcohol intake in which enrolled21, 30 and 33 cases, respectively. Histopathological changes of inflam-mation, fibrosis and fatty deposition in sections of liver biopsy fromthe patients were analyzed retrospectively.Results Liver biopsy assessment showed inflammation index andfibrosis index were significantly increased in patients with chronichepatitis B accompanied with alcohol intake. Fatty deposition in bothof patients with alcohol intake and with chronic hepatitis B accom-panied with alcohol intake were much more sever than those inpatients with simple chronic hepatitis B.Conclusion Alcohol-taking-in, especially the amount of alcohol-taking-in per day may cause hepatocellular damage to a certainextent, and had additional effects on deterioration of chronic hepati-tis B.

#150

The value of delta MELD in study the prognosis ofdecompensated cirrhosis patientsFEI LIU,1 WU JUN XIONG,1 YAN BING LIU,1

LAN ZHONG,1 ZHONG XIN ZHAO2

1Department of Gastroenterology, Shanghai East HospitalAffiliated to Tongji University, 2Department of Surgery,Shanghai East Hospital Affiliated to Tongji University

Aim To evaluate the prognostic value of delta MELD (model ofend stage liver disease) in decompensated cirrhosis patients.Methods The clinic datas of 97 decompensated cirrhosis patientswere analyzed retrospectively. Child-Turcotte-Pugh (CTP) score andMELD score were calculated for each patient following the originalformula on the admission day and after one month respectively. Thesubstraction of MELD scores between the first time and the secondtime mentioned above means delta MELD. The predictive power ofdelta MELD, MELD and CTP score was compared by using c-statistic. All patients were followed up at least 1 year.Results 10 of the 97 patients died in 3 months, whose delta MELD(3.23 ± 2.77) were higher than that of survivors (0.15 ± 0.39) (P <0.001). 18 of the 97 patients died in one year, whose delta MELD(3.00 ± 2.54) were higher than that of survivors (0.12 ± 0.42) (P <0.001).The area under receiver operating characteristic (ROC) curveof delta MELD was 0.835 compared with 0.782 (P < 0.01) of MELDand 0.745 of CTP score (P < 0.01) at 3 months; and they were 0.812,0.768 and 0.682 respectively (P < 0.01) at 1 year.Conclusions To evaluate the short and medium period prognosisof the patients with decompensated cirrhosis, the delta MELD isbetter than that of MELD and CTP as well, which is worth using inclinic widely.

#151

Association analysis of hepatitis B virus infection andpolymorphisms of HLA-A and B in Chinese HanpopulationSHU-MIN ZHAO,1 XIAO-HUI MIAO,1,1

WEN-SHENG XU,1 SHUN-LIANG TAN,2

CHENG-SONG ZHU3

1Department of infectious diseases, Changzheng hospital,Shanghai, China, 2Department of infectious diseases,The SecondPepole’s hospital, Changshu, Jiangsu province, 3Institute ofGenetics, Fudan University, Shanghai, China

HLA-A and B allele data of 70 Chinese Han subjects with HBV per-sistence after exposure in adult were compared with those of 120 withHBV clearance. Structural features of candidate allomorphs wereillustrated by similarity analysis.


Two HLA-A alleles, three HLA-B alleles and three HLA haplotypeswere significantly associated with outcomes of HBV infection afterexcluding the effect of the gender (Table 1). Similarity analysis indi-cated that allomorph A*0207 was characterized by cysteine at the 99th

amino acid residue (99 Cys) and B*4601 by 66 Lys and 69 Arg, whichare key residues of binding with peptide or TCR.In Chinese Han population, i) individuals positive for HLA haplo-type A*0207-B*4601 confer higher susceptibility for HBV persistentafter exposure, and the key amino acid residues substitution of HLA-A and B allomorphs involving in binding with peptide or TCR mightunderlie such association structurally; ii) A*3001-B*1302 positivelywhile A*1101-B*4001 were negatively associated with HBV clearance.

tissues correlated with the HBV markers in the veinal blood of thepregnant women. As likely as not, HBV was duplicated in fetal tissues,especially in fetal renal tissue, of the dead fetuses infected by HBVthrough maternal-fetal transmission.

#153

Hepatitis A seropositivity among patients with chronichepatitis B in ChinaCHENGYAN MENG,1 JIALING JIN,1

WENHONG ZHANG1

1Huashan Hospital, Fudan University, Shanghai, China

Background Hepatitis A and hepatitis B infections are prevalentthroughout Ascia. Patients infected with hepatitis B virus (HBV) havea higher morbidity and mortality if superinfected with hepatitis A.However, China being endemic for HAV, the prevalence of pre-existing antibodies against HAV due to subclinical exposure to thevirus may be high, therefore, vaccination should be considered on thebasis of Hepatitis A seropositivity surveillance.Methods Eight hundred and two patients with chronic HBV infec-tion seen at Huashan hospital at Shanghai in China, during the year2005 were tested for the presence of IgG anti-HAV antibody in theirsera (using a commercial ELISA kit).Results Due to the EPI starting of HAV vaccination in children inShanghai at the end of 1980s, the seroprevalence of patients at theage <20 was the highest as 91.4%. However, in the patients at age>20 years old, the seroprevalence of patients were lower and increasedwith age (64.8% in patients at 21–40 years old, 74.5% in patients at41–60 years old, 83.2% in patients >60 years old). The overall sero-prevalence among this population was 73.3%.Conclusion vaccination against HAV is not routinely requiredamong patients with chronic hepatitis B at age >60 in China as thereis a very high prevalence of pre-existing antibodies in these patients.However, HAV vaccination are still needed for the patients withchronic HBV infection who are susceptible to HAV, especially for thepatients in 21–40 years old who are with higher susceptible rate toHAV infection.

#154

The impact of HBeAg positive/negative and HBVDNAloads on the prediction of chronic severe hepatitis BQINGFENG SUN,1 DAOZHEN XU,2 YONG LV,3

JINGUO WU1

1Dep. of Infect. Dis.,The Third Affiliated Hospital,Wen ZhouMedical College, Zhejiang, China, 325200, 2Beijing DitanHospital, 3Fuzhou Infectious Hospital, Fujian

Objective To explore the impact of HBeAg positive/negative andHBV DNA loads on the prediction of chronic severe hepatitis B.Chronic severe hepatitis B is a severe disease with bad prognosis andhigh fatality rate, which is regarded as acute liver failure based onchronic hepatitis B.Methods 206 patiants with chronic severe hepatitis B hospitalizedin Beijing Ditan Hospital from Jul. 2002 to Dec. 2004 were analyzedretrospectively. HBeAg positive/negative, HBV DNA loads and otherfactors relating with prognosis were performed for univariate andmultivariate analysis. Result χ2 univariate analysis showed that therewas no significant difference of the prognosis between differentHBeAg groups (χ2 = 0.440, OR = 0.777, 95% CI 0.424–1.425, P =0.50).While there was significant difference of prognosis between dif-ferent HBV DNA loads groups, the prognosis of patients with lowerHBV DNA loads (HBV DNA <3 × 104 copies ·ml−1 in HBeAg neg-ative patients and <1 × 105 copies ·ml−1 in HBeAg positive patients =were better than that of patients with higher HBV DNA loads (χ2 =9.806, P = 0.002, OR = 3.055, 95% CI 1.554–6.007), the improving

Table 1 HLA class I alleles and haplotypes associated with consequence ofHBV infection

Allele or haplotype OR 95% CI Pfrequency (%) (Logistic

Persistence Clearanceregression)

HLA-A n = 62 n = 91 (excluding AHB)

A*0207 18.55 7.69 3.207 1.443∼7.128 0.0042A*1101 28.23 21.43 1.931 1.060∼3.519 0.0315

HLA-B n = 67 n = 111B*4601 15.67 7.21 2.984 1.394∼6.385 0.0049B*1302 3.73 10.36 0.315 0.113∼0.880 0.0275B*4001 20.90 10.81 1.956 1.000∼3.928 0.0501

Haplotype n = 60 n = 106 Z P (Z test)A*0207-B*4601 12.37 4.79 2.31 <0.05A*1101-B*4001 7.15 0.91 2.81 <0.05A*3001-B*1302 2.52 8.88 2.02 <0.05

n: Sample size of subjects successfully genotyped by sequencing-typing; Z:Statistic of Z test

#152

Preliminary study on the expression of HBcAg in ninedifferent organ tissues of the dead fetuses infected byHBV through maternal-fetal transmissionWEI LIU1

1The Second Hospital of Nanjing City

HBcAg in nine different organ tissues from 40 dead fetuses infectedby HBV through maternal-fetal transmission were detected byimmunocytochemistry (SP). HBV markers in the mothers’ veinalblood samples were detected by ELISA before delivery. Among theHBcAg positive rates in nine different organ tissues studied, pul-monic, hepatic, renal, and umbilical tissues occupied the first place,thymus, cardiac, lineal, and placentary tissues came the second, andcerebral tissue the last (p < 0.05). As considering with their mothers’blood samples tested, fetal pulmonic and umbilical tissues occupiedthe first place when the mothers showed HBVM+, renal tissue camethe second, and cerebral tissue the last (p < 0.05); fetal hepatic andrenal tissues occupied the first place when the mothers showedHBcAg+HbeAb+HbcAb, thymus, cardiac, pulmonic, lineal, umbili-cal, and placentary tissues came the second, and cerebral tissue thelast (p < 0.05); fetal thymus tissue occupied the first place when themothers showed HBcAg+HbeAg+HbcAb, cardiac, pulmonic, hepatic,lineal, renal, umbilical, and placentary tissues came the second, andcerebral tissue the last (p < 0.05). There were no significant differ-ences among the same fetal tissues (except thymus) of which themothers’ blood samples showed any of the three kinds of HBV posi-tive (p > 0.005).The distribution of HBV in different fetal organ


rate were 53.1% and 27.0% respectively. By the analysis of multi-variate logistic regression, 9 factors were screened and were showedgreat impact on chronic severe hepatitis B, such as, based on cirrho-sis, hepatorenal syndrome, hepatic encephalopathia, PTA <20%,TBil>513umolL−1, ALB <30gL−1, Cho <1.6mmolL−1, PLT <5 × 109 L−1,higher HBV DNA loads, and they were associated with poor prog-nosis. The coefficients of regression about HBV DNA loads were (B= 2.656, wald = 7.768, P = 0.005, EXP (B) = 14.235, 95.0% CI2.199–92.133).Conclusion HBV DNA loads is regarded as an important factoron prognosis of chronic severe hepatitis B, The lower the viral loads,the better the prognosis. But HBeAg positive/negative have no influ-ence on it.

#155

Quantitative detection of hepatitis B virus cccDNA inperipheral blood mononuclear cell and liver inpatients with chronic hepatitis BYONG HAO,1 XIAOHUI MIAO,1 KEKAI ZHAO1

1Shanghai Changzheng Hospital

Objectives To observe the existence and distribution of cccDNAin peripheral blood mononuclear cell (PBMC) and liver tissue inpatients with chronic hepatitis B.Methods Fifty PBMC samples and 50 liver puncture samples frompatients with chronic hepatitis B were investigated. Nuclear acid wasextracted and treated with Plasmid-Safe ATP-dependent DNase(PSAD), and further amplified by real-time fluorescent PCR withprimers spanning the two direct repeat region of HBV genome fordetermining HBV cccDNA.Results HBV cccDNA were undetectable in all PBMC samples,while both total HBV DNA and cccDNA were positive in all the liversamples. The total HBV DNA level in liver sample ranged from 3.19× 102 to 6.69 × 107 copies/ μg human genomic DNA, and the cccDNAranged from 7.32 × 100 to 6.51 × 106.The level of HBV DNA is 10.7folds to 9.2 × 105 folds as that of the cccDNA in the same liver sample.Conclusions HBV cccDNA was not detected in PBMCs, whichdidn’t support the view that HBV could replicate in PBMCs, whilecccDNA was detected in all liver puncture samples, and varied sig-nificantly in different patients, but was not positively related to thelevel of intracellular total HBV DNA.

#156

Development of a new HBsAg assay with improvedsensitivity to wild type and variant HBsAg for use onthe Ortho Clinical Diagnostics VITROS ECiQImmunodiagnostics SystemJ DIMENT,1 M VAN CLEVE,2 H TODD,2 C CHING,2

B HERRING,2 J ZHENG,1 P KILMARTIN1

1Ortho Clinical Diagnostics, 2Chiron Corporation

Objectives The need to improve the detection of HBsAg to over-come the effect of variants and mutants, as well as increase generalwild type HBsAg sensitivity, is evident from literature. The objectiveof this study was to increase the ability of the VITROS ECiQ assayto detect a range of HBsAg variants and to further increase the sen-sitivity of the test to wild type HBsAg.Materials and Methods Over 60 monoclonal antibodies werecharacterized on their binding patterns of mutant HBsAg and wildtype antigen. Improvements were assessed for detection of serocon-version panels and enhanced analytical sensitivity for wild type, sub-types and recombinant mutants. A range of artificial recombinantmutants was generated from the first loop (124–137) and second loop(137–147) regions that have the greatest effect on conformation. Finaltests employed a standard protocol VITROS≈ ECiQ System with

IntellicheckTM (90 tests per hour). Using the optimum reagents devel-oped an improved assay was developed ready for commercialization.Conclusions For HBsAg mutants in or around the first loop withrecombinant materials the sensitivity was improved by 11%–100%depending on the variant. For the second loop variants there was anincrease in sensitivity for all mutants, giving an average sensitivity of0.14ng/ml. All seroconversion panels could be detected at the sameor an earlier time-point compared with the existing assay. Precisionand specificity were state-of-the-art at 6% on a low level control and99.95% on donor samples.

#157

Treatment with peginterferon alfa-2a (40KD)(PEGASYS®) plus ribavirin (COPEGUS®) for just 24weeks is sufficient in HCV genotype 1 or 4 ‘super-responders’PETER FERENCI,1 FOR THE AUSTRIAN HEPATITISSTUDY GROUP2

1Medical University of Vienna, 2various hospitals in Austria

Retrospective analysis suggests shorter schedules may be suitable forgenotypes 1 or 4 patients with undetectable HCV RNA at week 4.Herein we report data from patients with undetectable HCV RNA atweek 4 of therapy participating in a prospective randomised multi-centre trial to customise therapy according to virological responses atweek 4 and 12.Methods Interferon-naïve adults with chronic hepatitis C genotype1 or 4 were enrolled. All received peginterferon alfa-2a (40KD)(PEGASYS®) 180 μg/week plus ribavirin (COPEGUS®) 1000/1200mg/day (Figure). At week 4, ‘super-responders’ (undetectableHCV RNA, <50IU/mL) were assigned to receive a further 20 weeks’therapy.Results To date, 94 of the106 ‘super-responders’ have completedtreatment and 62 (75% ITT; 86% PP) have achieved an SVR.Patients with an SVR had a lower median baseline HCV RNA thanrelapsers (P < 0.006).Conclusion 75% of genotype 1/4 ‘super-responders’ (HCV RNA<50IU/mL at week 4) achieved an SVR following treatment withpeginterferon alfa-2a (40KD) plus ribavirin for only 24 weeks. Thisrate is higher than the overall SVR rate seen in genotype 1/4 patientsin multinational randomized clinical trials treated with the sameregimen for 48 weeks. By the time of the congress, most of group Dpatients will have completed treatment.

#158

The Shanghai area patient with hepatitis B age and the illness number of different type casedistribution analyzesGUO-GUANG XU1, SHANMING WU1

1Shanghai Public Health Center

Our country is the virus hepatitis high popular area. In each typehepatitis, hepatitis B prevalence rate outstandingly.The Shanghai hos-pital for infectious diseases altogether admits each kind of virushepatitis patients 12,965 in 2000–2004, admits illness number everybetween 2424–2853 cases.The received remedy rate for patients withhepatitis B (noy including other combined virus hepatitis) in1986–2252 occupied 77.3%–82.5%. But virus hepatitis B + D orother were addicted to the liver virus mix infection still to account forthe certain proportion, reach to 7.3%–11.7%.The near 5 year hepati-tis A admits of illness number to reduce (50–156 cases) year by year,the year receive remedy rate 6.2% falls to 2.1% from 2001 to 2004.It was much low receive remedy rate last years. The receive rate ofhepatitis E keep between 5.1%–6.0% in near 5 year. Our hospital alto-


gether admits 10,358 patients with hepatitis B in 2000–2004 years,0–19 year old of age group year admits of illness number is 74–116case, accounts for various ages section prevalence rate to be lowest(3.0%–5.7%). The ≥60 year old of old age group prevalence rate isalso low, in11.2%–15.6%. But 20–39 year old is at present constitutesthe hepatitis B patient ‘s main crowd with 40–59 year old of two agessections, the year prevalence rate reach to 33.6%–42.8%,40.3%–48.7%, respectively. Admit every year, in our hospital2424–2853 patients with hepatitis B, but the patients with chronichepatitis B accounts for 47.7%–54.8%.The patients with cirrhosis ofillness number alsoreach to 21.5%–24.3%. The patients with acutehepatitis B is only 8.2%–16.9%. The hepatitis B mix other to beaddicted to the liver virus infection proportion to be by no means rarenear 5 years. It is admitted 177–334 cases every year.

#159

The prevalence of Genotype of hepatitis C virusbetween IVDU and non-IVDUKUAN FU LIAO,1,2 CHENG YUAN PENG,1,2

LAI SHIH WEI,1,3 PO HENG CHUANG,1,2

SHUEH CHO LAI,1,2 JEN WEI CHOU,1,2

CHIH PIN CHEN,1,2 WEN PANG SU1,2

1China Medical University Hospital, 2Division ofGastroenterology, 3Department of Family Medicine

Background and Aim The prevalence of genotype of hepatitis Cvirus (HCV) is an important test in clinical epidemiology as well asin the treatment of HCV carrier with combined interferon and rib-avirin. Response rate of treatment is related to the genotype of HCV.This study is to further investigate the different prevalence of HCVgenotype between IVDU and non-IVDU.Method From July. 2003 to June 2005, 479 cases of viral hepatitisC were included in this study, 281 cases were IVDU and 198 werenon-IVDU. Direct purification of HCV viral nucleic acids from serumwith QIAmp Viral RNA Mini Kit (QIAGEN); LightCycler RNAAmplification Kit SYBR Green I, Roche was used to detect the geno-type of HCV.Result The prevalence of HCV genotype 1 in IVDU group was40.56% (1a: 20.28%, 1b: 20.28%), genotype 2 59.44% (2a: 46.98%,2b: 12.46%); but in non-IVDU group genotype 1 occupied 61.12%(1a: 3.54%, 1b: 57.58%), genotype 2 was 36.87% (2a: 25.25%, 2b:11.62%).Conclusion The prevalence of genotype 1b of HCV was predom-inant in non-IVDU group, but 2a in IVDU group.

#160

Precore mutation in acute liver failure and its clinicalsignificance—an experience from New DelhiPREMASHIS KAR,1 ABDUL MALIK,2

BHUDEV CHANDRA DAS,3

SYED AKHTAR HUSSAIN4

1Maulana Azad Medical College and Lok Nayak Hospital, NewDelhi, India, 2PCR Hepatitis Lab, Maulana Azad MedicalCollege and LN Hospital, New Delhi, 3Institute for Cytology andPreventive Oncology (ICPO), Noida, UP, 4Department ofbiosciences, Jamia Milia Islamia University, New Delhi

Background It is believed that a HBV variant with precore stopmutation at nucleotide 1896 is associated with Acute Liver Failure(ALF) due to hepatitis B, as it interferes with synthesis of HBe-antigen. Clinically its biological importance is not well established.Aims and Objectives To evaluate the presence of precore muta-tions in cases of ALF and to assess their clinical significance.Materials and Methods A total of 103 cases of ALF over a period

of 3.5 years were studied for possible presence of precore mutations.They were evaluated on basis of history, examination, liver profile andserology (IgM-anti-HAV, HBsAg, IgM-anti-HBc, HBeAg, HBVDNA, anti-HCV and IgM antiHEV). Those who were positive forHBsAg, HBeAg and HBV DNA were subjected to Single StrandConformational Polymorphism (SSCP). Ligase Chain Reaction(LCR) was done in those who showed mobility shift in SSCP whichwas further confirmed by Direct Sequencing.Results 19 out of total 103 patients were positive for HBV DNA.5/19 (26%) showed evidence of mobility shift when subjected toSSCP. LCR also validated presence of mutants in 5/19 cases. Thefindings of SSCP and LCR were confirmed by Direct Sequencingwhich documented the presence of precore mutation with G to Aswitch at 1896 position in all the five cases. Only one case had evi-dence of mixed infection with wild and mutant form of virus. Therewere 11/14 (79%) deaths in the wild virus group whereas 5/5 (100%)mortality in mutant virus group.Conclusion The study suggested that the presence of precoremutant is associated with stormy course of ALF leading to high mortality.

#161

Plasma derived hepatitis B (pre-S + S) vaccine—evaluation of a potential newer therapeutic modalityin treatment of hepatitis BSHASHIDEEP SINGHAL,1 ARUN RAJAN,1

BHUDEV CHANDRA DAS,2 PREMASHIS KAR1

1Gastro. Division, Department of Medicine, MA Medical Collegeand LN Hospital, New Delhi, 2Institute for Cytology andPreventive Oncology (ICMR), Noida, UP

Background Partial efficacy of Interferon and development ofmutant strains with Lamivudine has initiated quest for newer thera-pies. Combination of pre-S and S protein has been found to be moreimmunogenic than S protein alone.Objectives Study was designed to evaluate plasma derived hepati-tis B vaccine (pre-S + S) in treatment of replicative carriers of HBV(HBsAg and HBV DNA positive) and to access efficacy by HBVDNA quantification (fall >50%) and supplement it by changes in T-cell counts and cytokine levels.Methods Seventy replicative carriers of HBV were randomised intotwo groups, with 35 patients receiving three doses of vaccine atmonthly intervals (cases) and the other group acting as controls. Allparameters were evaluated at initiation (M0) and repeated at 6months (M6).Results At M6 three of the vaccinated cases became HBsAg neg-ative and also developed anti-HbsAb, also 2 cases and 1 control devel-oped anti-HBe positivity. There was a significant decrease in HBVDNA levels and rise in CD4+T-cell counts in four and five casesrespectively. Mean values of HBV DNA were lower and also theCD4+T-cell counts were higher in cases as compared to controls.Mean values of IL-2, IL-12 and IFN-Y increased and IL-10 fell aftervaccination, suggestive of Th-1 type of response. When correlated,these values suggest that vaccination seems to bring about CD4+T-cell proliferation associated with a fall in HBV DNA levels.Conclusion This pilot study though time-bound: indicates that thedevelopment of strong T-cell response to HBV-related peptides byvaccination may help in overcoming the tolerance to chronic viralinfection.


#162

The virological response of serum HBV DNA inpatients with chronic hepatitis B after treatment withadefovir dipivixil or lamivudineFUHUA CHEN,1,2 MOBIN WAN,1 QIAN ZHANG,1

XUESONG LIANG1

1Department of Infectious Diseases of Changhai Hospital, SecondMilitary Medical University, Shanghai 2, 2Department ofInfectious Diseases of Guizhou Provincial Corps Hospital,Chinese People, s Armed Polic

Background Currently, FDA-aproved regimens for the treatmentof CHB include interferon-alpha2b, peginterferon-alpha2a, lamivu-dine, adefovir dipivoxil and entecavir. These treatment are subopti-mal. In the search for improved therapies, we have carried this clinicaltrial.Objective To compare the serum virological response and end-of-dose failure after treatment with adefovir dipivixil (ADV) or lamivu-dine in patients with chronic hepatitis B infection.Methods 52 patients were divided into two groups randomly, andone group was given ADV 10mg1/d, the other was given 3TC 100mg/d. The treatment last 52 weeks, and followed for 12 weeksafter treatment. Serum HBV RNA level of all patients were examinedrespectively before and 4w, 12w, 24w, 36w, 48w, 52w during thetreatment and 12w after therapy.Results The serum HBV DNA response rate of the group treatedwith ADV was lower than that of 3TC group at different time duringtherapy. But only at 12w during therapy, the HBV DNA responserate between the two group had significant deviation (X2 = 5.023, P< 0.05). The end-of-dose failure of ADV group was lower than thatof the 3TC group (21.43% vs 31.58%), and the end-of-dose failureof patient with HBeAg(+) of the two group was 10.00% vs37.49%respectively, and that of the patient with HBeAg(−) was 50.00%,9.091% (P > 0.05).Conclusion Both ADV and 3TC can effectively suppress the serumHBV DNA in patient with chronic hepatitis B infection. But the earlyresponse rate and end-of-dose failure rate of the two group was sig-nificantly different.

#163

Effects of fatty liver and related factors on the efficacyof combination antiviral therapy in patients withchronic hepatitis CJIANWU YU,1 SHUCHEN LI,2 GUIQIANG WANG3

1Department of infectious diseases,The Second AffiliatedHospital, Harbin Medical University, 2Department of infectiousdiseases,The Second Affiliated Hospital, Harbin MedicalUniversity, 3Department of infectious diseases,The First Hospital,PeiKing University

Objective To investigate whether the fatty liver and related factorshave impact on the efficacy in chronic hepatitis C treated with pegin-terferon and ribavirin, and the associations between HCV genotyp-ing and fatty liver.Methods 98 patients received subcutaneously 180 μg peginterfer-onα-2a once a week plus ribavirin.The body mass index (BMI), waistto hip ratio (WHR) and HOMA-IR were calculated.Results The prevalence of fatty liver was 10.5%; 11.4%; 38.5% inpatients infected HCV genotype 1, 2, 3, respectively, which suggestedthat the distribution of fatty liver in different HCV genotypes wasimbalanced (χ2 = 6.758, P = 0.034). In univariate analysis, the effi-cacy of combination therapy was significantly associated with BMI (P= 0.011),WHR (P = 0.024), the levels of plasma insulin (P = 0.001),genotype (P = 0.036), presence of fatty liver (P = 0.028), treatmentdosage and duration (P = 0.012) and HOMA-IR (P = 0.002). With

Binary logistic regression analysis, the plasma insulin levels andHOMA-IR showed independent predictors to the efficacy of antivi-ral therapy.Conclusion The level of plasma insulin and HOMA-IR were inde-pendent factors for predicting effect of antiviral therapy.

#164

Prediction of the prognosis in patients with acute onchronic hepatitis using MELD scoring systemJIANWU YU,1 SHUCHEN LI,1 GUIQIANG WANG2

1Department of Infectious Diseases,The Second AffiliatedHospital, Harbin Medical University, 2Department of infectiousdiseases, the first hospital of Peiking university, Peiking

Objective To predict prognosis in patients with acute on chronichepatitis (AOCH) by MELD (model for end-stage liver disease,MELD) scoring system and study the effects of age, sex, etiology, lowserum sodium, persistent ascites on MELD.Methods MELD scores of 300 patients with AOCH were calcu-lated according to the original formula. The influential factors ofMELD were also discussed.Results The concordance (c) statistic of 3-month’s mortality was0.782. Univariate analysis showed that mortality was significantlyrelated to age (P = 0.047), etiology (P = 0.039), serum sodium (P =0.029) and ascites (P = 0.031) for patients with MELD scores 20–29.In multivariate analysis, in patients with MELD scores 20–29, age (P= 0.012), etiology (P = 0.024), serum sodium (P = 0.005) and ascites(P = 0.017) were independent predictors of mortality; for MELDscores above 30, only MELD score (P = 0.015) was independent predictive.Conclusions In the group with MELD score 20–29, the factorsincluding age, etiology, presence of low serum sodium and persistentascites may influence MELD scoring system.The MELD score is thedecisive predictor the prognosis of patients with AOCH when MELDscore is over 30.

#165

Inhibition of hepatitis B virus gene expression byRNA-cleaving 10–23 DNAzymes with differentsubstrate-binding arms in HepG2.2.15 cellsJIANER WO,1,2 WEI HOU,1,2 MINWEI LI,1,2

KEZHOU LIU1,2

1Key Laboratory of Infectious Diseases, Ministry of PublicHealth of China, 2Institute of Infectious Diseases, 1st AffiliatedHospital, College of Medicine, Zhejiang University

Inhibition of hepatitis B virus gene expression byRNA-cleaving 10–23 DNAzymes with differentsubstrate-binding arms in HepG2.2.15 cellsJIANER WO,* WEI HOU, MINWEI LI, KEZHOU LIU*Key Laboratory of Infectious Diseases, Ministry of PublicHealth of China; Institute of Infectious Diseases, First AffiliatedHospital, College of Medicine, Zhejiang University

RNA-cleaving 10–23 DNAzyme has been used successfully to sup-press the expression of viral or endogenous target genes in vitro andin vivo.The cleavage efficiency of 10–23 DNAzyme is remarkably sen-sitive to the length and composition of the substrate-binding arms. Inorder to explore the inhibition effects of 10–23 DNAzymes with dif-ferent substrate-binding arms targeting at HBV gene, in this study,various 10–23 DNAzymes specific to HBV S gene ORF A157UG andHBV C gene ORF A1816UG were transfected into HepG2.2.15 cells,a stable HBV producing cell line, respectively. HBsAg and HBeAgsecretions into culture media were detected by RIA and HBV DNA


levels were analyzed by real-time PCR. MTS assays were performedto evaluate cytotoxicity accordingly. Results indicated that HBsAgand HBeAg expressions were reduced by various DNAzymes respec-tively after transfection. The antiviral effects of DNAzymes were stillapparent 72h post-transfection. DrzBS-9 and DrzBC-9 could signif-icantly inhibit HBV S gene and C gene, as HBsAg and HBeAg werereduced 95% and 92% 48h post-transfection at the dose of 2.5 μM,respectively. There were no evident inhibition effects on HBV DNAand no evident cytotoxic effects of these DNAzymes. Therefore,10–23 DNAzymes with different substrate-binding arms targeting atHBV S and C gene all possessed the specific inhibition effects inHepG2.2.15 cells, DrzBS-9 and DrzBC-9 with longer substrate-binding arms were most potent, which would be a promising appli-cation in the treatment of HBV infection.

#166

Inhibition of hepatitis C virus gene expression by10–23 DNAzymesJIANER WO,1,1 WEI HOU,1,2 MINWEI LI,1,2

KEZHOU LIU,1,2 DENNIN REINHARD3

1Institute of Infectious Diseases, 1st Affiliated Hospital, College ofMedicine, Zhejiang University, 2Key Laboratory of InfectiousDiseases, Ministry of Public Health of China, 3Institut furMedizinische Mikrobiologie und Hygiene, Universitat zuLubeck, Lubeck, Germany

Hepatitis C virus (HCV) infection is a leading cause of chronichepatitis, cirrhosis and hepatocellular carcinoma (HCC). Presentantiviral therapy is still suboptimal and noval therapeutic approachesare an important objective. DNAzymes can be designed to cleavetarget RNA sequences specifically and show promise for the treat-ment of HCV infection. Demonstration of inhibitory effects of HCVgene expression by RNA-cleaving DNAzymes has been the aim ofthis investigation.We designed three DNAzymes with 10–23 catalyticmotif, DrzHCV-7, DrzHCV-8 and DrzHCV-9, to cleave target sitesat nucleotides 342 in the HCV open reading frame (ORF). In vitro,DrzHCV-7, DrzHCV-8 and DrzHCV-9 all could cleave target HCVRNAs at their specific target sites. Enhanced green fluorescent protein(EGFP) as a kind of report molecule, when each of the DNAzymeswere cotransfected in HepG2 cells with HCVCORE (BT1–172)-EGFP fusion expression plasmid containing truncated HCVCORE-EGFP fusion gene, DrzHCV-7, DrzHCV-8 and DrzHCV-9 allreduced the HCVCORE (BT1–172)-EGFP expression, HCVCORE(BT1–172)-EGFP mRNA levels were also reduced dramatically bythe DNAzymes.The result suggest that DNAzyme possessed the spe-cific inhibitory effects of HCV gene expression, which is useful todevelop strategies for gene therapy of liver diseases caused by HCV.

#167

Specific inhibition of hepatitis B virus X geneexpression by 10–23 DNAzymesWEI HOU,1,2 QIN NI,1,2 JIANER WO,1,2 MINWEI LI,1,2

KEZHOU LIU1,2

1Key Laboratory of Infectious Diseases, Ministry of PublicHealth of China, 2Institute of Infectious Diseases, 1st AffiliatedHospital, College of Medicine, Zhejiang University

The X protein (HBx) of human hepatitis B virus (HBV) is a tran-scriptional activator protein.The HBx protein plays an important rolein viral replication in HBV infected cells and the liver diseases includ-ing hepatitis, cirrhosis, and hepatocellular carcinoma (HCC). There-fore, the repression of HBx gene expression by 10–23 DNAzymesmight be a good way to inhibit HBV replication and cure HBV-relatedliver diseases. We designed three 10–23 DNAzymes with differentsubstrate-recognition domains. When each of the 10–23 DNAzymes

were cotransfected into human AD293 cells with HBx-EGFP expres-sion plasmid, they could all reduced the level of HBx mRNA. TheHBx-EGFP protein was also reduced dramatically by the10–23DNAzymes. These results suggest that the 10–23 DNAzymes mightbe used for the gene therapy of liver diseases caused by HBV.

#168

Phylogenetic, virological and clinical characteristicsof hepatitis B virus with TCC at codon 15 of theprecore regionHLY CHAN,1 CH TSE,1 KS LEUNG,2 KH LEE,2

SKW TSUI,3 VWS WONG,1 JJY SUNG1

1Institute of Digestive Disease,The Chinese University of Hong Kong, 2Department of Computer Sciences andEngineering,The Chinese University of Hong Kong,3Department of Biochemistry,The Chinese University of Hong Kong

Hepatitis B virus (HBV) with T-1856 of the precore region is alwaysassociated with C-1858 (i.e. TCC at nucleotides 1856–1858), and itis only reported in genotype C HBV.We aimed to investigate the phy-logenetic, virological and clinical characteristic of HBV isolatesbearing TCC at nucleotides 1856–1858.We have previously reportedthe presence of 2 major subgroups in genotypes C HBV, namelyHBV/Cs (Southeast Asia) and HBV/Ce (Far East).We have designeda novel 5′nuclease technology based on the nucleotide polymorphism(C/A) at nucleotide 2733 to differentiate the 2 genotype C HBV sub-groups. The mutations at basal core promoter and precore regionswere analyzed by direct sequencing. Among 214 genotype C HBVinfected patients, 31% had TCC, 37% had CCC, 3% had CTC and29% had CCT at nucleotides 1856–1858. All except one HBV strainwith TCC at nucleotides 1856–1858 belonged to subgroup HBV/Csreported only in Hong Kong, Guangzhou and Vietnam. HBV withTCC at nucleotides 1856–1858 was associated with G1898A muta-tion (64%). Patients with HBV harboring TCC had more liver cir-rhosis than those with CCC (18% versus 5%, p = 0.008), and hadmore positive HBeAg (58% versus 36%, p = 0.01), higher medianALT levels (65 IU/l versus 49 IU/l, p = 0.006) as compared to thosewith CCT. In summary, we reported that HBV with TCC atnucleotides 1856–1858 of the precore region might represent a spe-cific HBV strain associated with more aggressive liver disease thanother genotype C HBV.

#169

Pegylated interferon alfa-2b plus ribavirin in the re-treatment of interferon-ribavirin non responder patientsGLORIA TALIANI,1 GIULIA GEMIGNANI,2

CARLO FERRARI,3 ANTONIO ACETI,1

DARIO BARTOLOZZI,4 PIER LUIGI BLANC,4

MARCO CAPANNI,2 FRANCESCO ESPERTI,5

PAOLO FORTE,4 VINCENZO GUADAGNINO,6

TERENZIO MARI,7 NICOLETTA MARINO,4

STEFANO MILANI,2 CATERINA PASQUAZZI,1

DANILO TACCONI,8 MARIO TOTI,9

ANNA LINDA ZIGNEGO,2 LUCA MESSERINI,2

ELISA BILIOTTI,1 TOMMASO STROFFOLINI7

1University “La Sapienza” Rome, 2University of Florence,3Azienda Ospedaliera Parma, 4Azienda Ospedaliera Firenze,5Azienda Ospedaliera Pistoia, 6University of Catanzaro,7Ospedale S Giacomo Roma, 8Azienda Ospedaliera Arezzo,9Azienda Ospedaliera Grosseto


Background and Aims Inadequate data are available aboutretreatment of non responders to interferon (IFN) and ribavirin.Thisstudy evaluated the efficacy and tolerability of a 48-week therapy withpeginterferon alfa-2b plus high dose ribavirin in patients who havefailed to respond to the combination. Treatment up to 48 weeks alsoin patients who failed to clear HCV-RNA by week 24 was also evaluated.Patients and Methods One-hundred-forty-one patients who pre-viously did not respond to IFN and ribavirin, 85% with henotype1/4infection, 52% with high viral load (>800.000 IU/ml), 22% with cir-rhosis, were retreated with peginterferon alfa-2b 1.5 μg/Kg and rib-avirin 1000–1200mg/day for 48 weeks and followed-up for 24 weeks.Results By intent-to-treat analysis, 20% of patients achieved a sus-tained virological response (SVR). SVR of genotype-1 patients was19%. Independent predictors of SVR were low γGT levels (O.R. 22.9;C.I. 95% = 6.6–79.9) and low viral load (O.R. 3.8; C.I. 95%1.1–12.7).Twelve (23%) out of 51 patients who were HCV-RNA pos-itive after 24 weeks of therapy achieved a late virological response(after week 24) and 5 (10%) of them, all with genotype 1, achievedan SVR. Genotype was not associated neither with response (p = 0.2),neither with early response (p = 0.3).Conclusions Retreatment with peginterferon-alfa-2b and ribavirinof multi-experienced and difficult-to-treat non responder patientsproduced a very promising SVR. Accurate selection of patients, suchas those with low viral load and low γGT levels, and prolongation oftherapy beyond 24 weeks also in HCV-RNA positive patients mayfurther increase the rate of SVR.

#170

Hepatitis B virus X gene and cyanotoxins promoteaflatoxin B1-induced hepatotumorigenesis in miceSHUN-ZHANG YU,1 MIN LIAN,1 YING LIU,1

GENG-SUN QIAN,2 SHU-GUANG WAN2

1Institute of Prebventive Medicine, Fudan University, 2ShanghaiCancer Institute, Shanghai Jiao Tong University

Aim To assess the combinative role of aflatoxin B1 (AFB1),cyanobacterial toxins (cyanotoxins), and hepatitis B virus x gene(HBx) in hepatotumorigenicity.Methods One-week-old animals carrying HBx and their wild-typelittermates were i.p. injected with either single-dose AFB1 (6mg/kgb.w.), repeated-dose cyanotoxins (microcystin-LR or nodularin,10 μg/kg b.w. once a week for 15 weeks), DMSO (vehicle control)alone, or AFB1 followed by cyanotoxins a week later, and were sacri-ficed at 24 weeks and 52 weeks post-treatment.Results AFB1 induced liver tumors in 13 of 29 (44.8%) transgenicmice at 52 weeks, significantly more frequent than in wild-type mice(13.3%). This significant difference was not shown in the 24-weekstudy. Compared with AFB1 exposure alone, MC-LR and nodularinyielded approximately 4-fold and 7-fold increases in the incidence ofAFB1-induced liver tumors in wild-type animals at 24 weeks, respec-tively. HBx did not further elevate the risk associated with co-exposure to AFB1 and cyanotoxins. With the exception of a MC-LR-dosed HBx−/− mouse, no liver tumor was observed in mice treatedwith cyanotoxins alone at 24 weeks. Neither DMSO-treated trans-genic mice nor their wild-type littermates had pathologic alterationsrelevant to hepatotumorigenesis in even up to 52 weeks.Conclusion HBx and nodularin promoted the development ofAFB1-induced liver tumors. Co-exposure to AFB1 and MC-LRtrended to elevate the risk of liver tumors at 24 weeks relative to expo-sure to one of them.The combinative effect of AFB1, cyanotoxins andHBx on hepatotumorigenesis was weak at 24 weeks.

#171

Does insulin resistance occur in patients with chronichepatitis C with genotype 3?AJAY DUSEJA,1 YOGESH CHAWLA,1

RADHAKRISHAN DHIMAN,1 SANJEEV SHARMA,1

KIRAN KUMAR T,1 CHANDERSHEKHAR REDDY,1

ASHIM DAS,2 SANJAY BHADADA,3 R SIALY,3

ANIL BHANSALI3

1Department of Hepatology, PGIMER, Chandigarh, India,2Department of Histopathology, PGIMER, Chandigarh, India,3Department of Endocrinology, PGIMER, Chandigarh, India

Background & Objective HCV genotype is one of the determi-nants for hepatic steatosis and insulin resistance in patients withchronic hepatitis C (CHC). Objective of study was to determine theinsulin resistance (IR) in Indian patients with CHC with predomi-nant genotype 3.Methodology Forty-five (men 40, mean age 38.9 ± 11.4 years,mean BMI 24.7 ± 3.5kg/m2) non-diabetic, biopsy proven patients ofHCV related chronic hepatitis without cirrhosis were included.Fasting serum insulin levels were determined by radioimmunoassayand insulin resistance by the homeostasis model of assessment(HOMA). The results were compared with 38 (men 25, mean age38.7 ± 10.9 years, mean BMI 26.9 ± 3.9kg/m2) biopsy proven patientsof nonalcoholic fatty liver disease (NAFLD) and 25 (men 20, meanage 36.9 ± 12.9 years, mean BMI 23.6 ± 2.7kg/m2) age and BMImatched healthy volunteers (HV). Serum tumor necrosis factor–alpha(TNF-α) levels and C-peptide (ELISA) levels were also measured inall 3 groups.Results Patients with CHC (genotype 3 = 31, genotype 1 = 11) hada higher fasting insulin (174.7 ± 210.1 Vs 37.8 ± 34.2pmol/L) andHOMA-IR (8.2 ± 6.6 Vs 1.4 ± 1.2) values in comparison to HV (p< 0.001). Higher number of NAFLD patients (97%) had IR in com-parison to CHC patients (65%) (p < 0.001). Even though the levelswere higher in patients with genotype 1, there was no difference ininsulin (209.1 ± 254.4 Vs 109.7 ± 5.3 p = not significant) andHOMA–IR (7.5 ± 9.2 Vs 3.8 ± 1.6, p = not significant) levels amongpatients of genotype 1 and genotype 3.Conclusion Insulin resistance is common in non-diabetic, non-cirrhotic Indian patients with chronic hepatitis C with genotype 3.

#172

B7-H1 expression is up-regulated in peripheral bloodCD14+ monocytes of patients with chronic hepatitis Bvirus infection, which correlates with higher serumIL-10 levelsLEI GENG,1 SHUSEN ZHENG1

1First Affiliated Hospital, Zhejiang University School of medicine

Chronicity in Hepatitis B virus infection is maintained by increasedtype-2 T helper cell response, possibly due to increased interleukin10 productions. B7-H1 can negatively regulate T cell responses via itsreceptor, programmed death 1. The ligation of B7-H1 to T cells canresult in the preferential secretion of interleukin 10. In this study, weinvestigated whether there was an up-regulated expression of B7-H1in peripheral blood mononuclear cells in patients chronically infectedby HBV and further explored the correlation between B7-H1 expres-sion and serum interleukin 2, interferon-γ, interleukin 10, alanineaminotransferase levels and viral load. Fifty-five patients with chronicHBV infection and twenty healthy controls were enrolled in thepresent study.The results showed that in patients with chronic hepati-tis B CD14+ monocytes, but not CD3+ and CD19+ cells had a sig-nificantly increased expression of B7-H1 compared to healthycontrols, which positively correlates with serum interleukin 10 levelsand the present of HBeAg and negatively correlates with serum


alanine aminotransferase levels. In conclusion, Chronic HBV patientsharbor an increased B7-H1 expression in CD14+ monocytes com-pared with controls, which may be responsible for the increasedserum IL-10 levels.This might be an important way by which hepati-tis B virus evades an adequate immune response, leading to viral per-sistence and disease chronicity.

#173

PI3K/DNp63 pathway is involved in HCV infected B-cells transformationCLARA BALSANO1,2

1University of L’Aquila, 2Fondazione Andrea Cesalpino

HCV core protein has been shown to deregulate cell growth and pro-grammed cell death in hepatoma cells, but only few information areavailable about its possible role in B lymphocytes. The aim of ourwork was to analyze the biological activity of HCV core protein on Bcells, regarding cell proliferation and apoptosis. We established Wil2-ns and Ramos B cells stable expressing HCV core protein.Thymidine incorporation analysis, as well as PCNA, activated-ERKand FACS analysis demonstrates that HCV core induces an increaseof growth in both cell lines. TUNEL cytofluorimetric analysis indi-cates a low rate of apoptosis (5%) in the same cells. Hence, we haveinvestigated whether, in B lymphoma cells, HCV core protein shouldbe able to alter p53 family functions. HCV core protein, in our model,modifies cell growth homeostasis by the induction of DNp63 associ-ated to a down-regulation of DNp73 mRNAs.

#174

Entecavir (ETV) is superior to lamivudine (LVD) for the treatment of chronic hepatitis B (CHB):results of a phase 3 Chinese study (ETV-023) innucleoside-naïve patientsGUANGBI YAO,1 CHENGWEI CHEN,2 WEILUN LU,3

HONG REN,4 DEMING TAN,5 YUMING WANG,6

DAOZHENG XU,7 JESSICA LIU,8 DONG XU,9

LAURIE MACDONALD,9 MICHAEL JIANG8

1Shanghai Jing An Central Hospital, Shanghai, China,2Shanghai Liver Disease Center Of Nangjing Military Area,Shanghai, China, 3No. 3 Hospital Affiliated to ZhongshanMedical University, Guangzhou, China, 4No. 2 HospitalAffiliated to Chongqing Medical University, Chongqing, China,5Infectious Disease Dept. of Xiang Ya Hospital, Changsha,China, 6Xinan Hospital, Chongqing, China, 7Beijing Di TanHospital, Beijing, China, 8Bristol-Myers Squibb Company,Shanghai, China, 9Bristol-Myers Squibb Company,Wallingford,CT, USA

Background Entecavir is a potent inhibitor of hepatitis B virus(HBV) with proven clinical efficacy. A randomized, double-blind,multi-center study was conducted in China in nucleoside-naïvepatients with CHB.Methods 519 patients were treated with ETV 0.5mg (n = 258) orLVD 100mg (n = 261) for at least 52 weeks. The primary endpointwas a composite endpoint of HBV DNA <0.7MEq/mL by bDNAassay and ALT <1.25 × ULN at Week 48.Results Most patients were male (82%), the mean age was 30years, and 86% were HBeAg(+). Results for primary and key sec-ondary endpoints are presented below:

Overall incidence of adverse events (AEs) was comparable: 60% ofETV patients and 56% of LVD patients reported adverse events; 3%of ETV patients and 5% of LVD patients reported serious AEs.Conclusions ETV achieves superior virologic and biochemicalimprovements in nucleoside-naïve patients with CHB with a safetyprofile comparable to LVD.

#175

Modeling clinical benefits of suppressing viralreplication in chronic hepatitis B (CHB) patients inChina: a number-needed-to-treat (NNT) analysis ofentecavir and lamivudine based on a phase IIIrandomized clinical trialUCHENNA ILOEJE,1 YONG YUAN,2 JOEL HAY,3

GUANG BI YAO4

1Bristol-Myers Squibb Pharmaceutical Research Institute,Wallingford, CT, USA, 2Bristol-Myers Squibb PharmaceuticalResearch Institute, Plainsboro, New Jersey, USA, 3University ofSouthern California, Los Angeles, Californina, USA, 4ClinicalImmunology Research Center, Jin An District Hospital &Shanghai Fudan University

Objectives Approximately 112 million Chinese have CHB-infection. Entecavir is a recently approved therapeutic option forCHB infection. We estimate the number of patients who would needto be treated with entecavir instead of lamivudine to prevent one livercomplication using data from Chinese patients.Methods Empirically observed data on progression to HCC andcirrhosis based on the level of viral replication from a large scale epi-demiology study was matched to data from BMS study AI-463023,a randomized, double blind, phase III clinical trial of 519 CHBpatients (males (82%); HBeAg+ (87%); mean age 30) in China. Tenyear projections of liver complications were based on a hypotheticalcohort of 1000 patients. The NNT was calculated as the reciprocalof the projected absolute risk reduction at 10 years, and its 95% con-fidence interval was constructed to estimate the uncertainty in theNNT.Results Out of these hypothethical patients, projected number ofevents were: entecavir 19 HCC, and 47 cirrhosis; lamivudine 71 HCCand 132 cirrhosis. The projected ten-year risks of HCC and Cirrho-

Primary And Key Secondary Endpoints At Week 48

ETV 0.5 mg LVD 100 mg p-valuen = 258 n = 261(treated) (treated)

HBV DNA <0.7 MEq/mL 90% 67% <0.0001and ALT <1.25 × ULN

HBV DNA Mean Change −5.90 −4.33 <0.0001From Baseline by PCR(log10 copies/mL)

HBV DNA <300 copies/mL 76% 43% <0.0001

ALT Normalization 90% 78% 0.0003(<1 × ULN)

HBeAg Seroconversion 15% 18% 0.39(n, HBeAg+ at baseline) (n = 225) (n = 221)

HBV DNA Mean Change From Baseline (log10 copies/mL) (*p < 0.0001)

Treatment Baseline Week 12 Week 24 Week 36 Week 48

ETV 0.5 mg 8.64 −5.07* −5.45* −5.56* −5.90*LVD 100 mg 8.48 −4.53 −4.68 −4.51 −4.33


sis with entecavir therapy were 1.9% and 4.7%, respectively, vs. 7.1%and 13.2% with lamivudine therapy. The projected risk of liver com-plication was 6.1% for entecavir vs. 18.3% for lamivudine, resultingin an NNT of 8 patients (95% CI: 7–11) needing to be treated withentecavir instead of lamivudine to prevent one cirrhosis or HCC in10 years.Conclusions The NNT to prevent one liver complication pro-jected from this model suggests a favorable benefit profile for entecavir.

#176

Entecavir (ETV) results in early viral load reductionin chronic hepatitis B (CHB) patients who have failedlamivudine (LVD) therapy: a randomized,placebo-controlled study in China (study ETV-056)GUANGBI YAO,1 X ZHOU,2 DAOZHENG XU,3

B WANG,4 HONG REN,5 MICHAEL JIANG,6

JESSICA LIU,6 DONG XU,7 LAURIE MACDONALD7

1Shanghai Jing An Central Hospital, Shanghai, China, 2Rui JinHospital Affiliated to Shanghai No. 2 Medical University,Shanghai, China, 3Beijing Di Tan Hospital, Beijing, China,4Beijing Friendship Hospital Affiliated to Capital MedicalUniversity, Beijing, China, 5No. 2 Hospital Affiliated toChongqing Medical University, Chongqing, China, 6Bristol-Myers Squibb Company, Shanghai, China, 7Bristol-MyersSquibb Company,Wallingford, CT, USA

Background ETV is a potent inhibitor of wild-type and LVD-resistant hepatitis B virus. A randomized, multi-center study was con-ducted in China in patients who had failed LVD.Methods Patients received blinded treatment with ETV 1.0mg (n= 116) or placebo (n = 29) for 12 weeks. All patients then receivedopen-label ETV for 36 weeks. The primary endpoint was the meanchange in HBV DNA from baseline at Week 12.Results The mean change in HBV DNA at Week 12 was −4.30 log10 copies/mL for ETV and −0.15 log10 copies/mL for placebo(p < 0.0001). ETV produced rapid reductions in HBV DNA.

After 48 weeks of ETV treatment, 27% of patients had undetectableHBV DNA (<300 copies/mL) and 85% had achieved ALT normal-ization. ETV 1.0mg for 48 weeks was generally well-tolerated.Conclusions ETV is a potent antiviral that achieves rapid and pro-found reductions in HBV DNA in patients who failed LVD.

#177

Efficacy and safety of entecavir (ETV) and lamivudine(LVD) in compensated, cirrhotic patients with chronichepatitis BTING-TSUNG CHANG,1 YOU-CHEN CHAO,2

SHUN-SHENG WU,3 CHEE KIA TAN,4

MOHAMED ROSMAWATI,5 ALICE LEE,6

JACOB GEORGE,7 HELENA BRETT-SMITH,8

RICHARD ZINK,8 BOON-LEONG NEO9

1National Cheng Kung University Medical College,Tainan,Taiwan, 2Tri-Service General Hospital,Taipei,Taiwan,3Changhua Christian Hospital, Changhua,Taiwan, 4SingaporeGeneral Hospital, Singapore, 5University Malaya MedicalCentre, Kuala Lumpur, Malaysia, 6Concord RepatriationGeneral Hospital, Concord, NSW, Australia, 7Storr Liver Unit,University of Sydney,Westmead Hospital,Westmead, NSW,Australia, 8Bristol-Myers Squibb Pharmaceutical ResearchInstitute,Wallingford, CT, USA, 9Bristol-Myers SquibbPharmaceutical Research Institute, Singapore

Background Antivirals have been shown to slow disease progres-sion in CHB patients with compensated cirrhosis. This subgroupanalysis assessed 48-week treatment responses in patients with base-line cirrhosis (Knodell fibrosis score of 4).Methods Studies ETV-022 and ETV-027 enrolled treatment-naïve, HBeAg(+) and HBeAg(−) patients, respectively. ETV-026enrolled LVD-refractory, HBeAg(+) patients. Liver biopsies were per-formed before study entry and after 48-weeks’ therapy.Results Approximately 8% of patients had baseline cirrhosis andwere distributed evenly between treatment groups. Cirrhotic patientson ETV were more likely than LVD-patients to have histologicimprovement, ALT normalization and HBVDNA <300c/mL. Resultsfor nucleoside-naïve patients according to baseline cirrhosis status arepresented below.In the LVD-refractory population, more ETV- vs LVD-treated cir-rhotic patients achieved histologic improvement (50% vs 22%),HBVDNA <300c/mL (21% vs 0%) and ALT normalization (50% vs11%).Conclusions Overall, responses were consistent between patientswith cirrhosis and total populations for HBeAg(+), HBeAg(−) nucle-oside-naïve and HBeAg(+) LVD-refractory patients.

Week Mean change in HBV DNA (log10 copies/mL)

ETV (n = 116)

2 −2.654 −3.218 −3.96

12 −4.3024 −4.8948 −5.08

Efficacy Endpoints At Week-48 Among Efficacy Endpoints At Week-48 Among Treatment-Naïve Patients Treatment-Naïve Patients

Week-48 HBeAg(+) HBeAg(−)

ETV LVD ETV LVD

Cirrhotic Total patient Cirrhotic Total patient Cirrhotic Total patient Cirrhotic Total patientpatients population patients population patients population patients populationn = 25 n = 354 n = 27 n = 355 n = 19 n = 325 n = 28 n = 313

Histologic 76% 72% 67% 62% 74% 70% 54% 61%Improvement*

HBV DNA 96% 67% 59% 36% 95% 90% 57% 72%<300copies/mL

ALT < 1.0 × ULN 60% 68% 52% 60% 79% 78% 57% 71%

*Histologic Improvement: ≥2 point improvement in Knodell Necroinflammatory Score with no worsening of Knodell fibrosis score at Week 48.


#178

Continued virologic and biochemical improvementthrough 96 weeks of entecavir treatment in HBeAg(-)chronic hepatitis B patients (Study ETV-027)CHING-LUNG LAI,1 TING-TSUNG CHANG,2

YOU-CHEN CHAO,3 TAWESAK TANWANDEE,4

SATAWAT THONGSAWAT,5 SHOU-DONG LEE,6

PETER ANGUS,7 RICHARD ZINK,8 JIN ZHU,8

HELENA BRETT-SMITH,8 BOON-LEONG NEO9

1Queen Mary Hospital, Hong Kong, China, 2National ChengKung University Medical College,Tainan,Taiwan, 3Tri-ServiceGeneral Hospital,Taipei,Taiwan, 4Siriraj Hospital, MahidolUniversity, Bangkok,Thailand, 5Maharajnakorn Chiang MaiHospital, Chiang Mai,Thailand, 6Taipei Veterans GeneralHospital,Taipei,Taiwan, 7Austin & Repatriation MedicalCentre,West Heidelberg,VIC, Australia, 8Bristol-Myers SquibbPharmaceutical Research Institute,Wallingford, CT, USA

Background Entecavir (ETV) demonstrated superior virologicand histologic benefit over lamivudine (LVD) after 48 weeks oftherapy in HBeAg(−) CHB patients. The efficacy and safety datathrough 96 weeks of treatment are reported here.Methods Patients were randomized 1 :1 to receive either ETV 0.5mg (n = 325) or LVD 100mg (n = 313) for 48 weeks. Respon-ders (HBV DNA <0.7MEq/mL by bDNA assay and ALT < 1.25 ×ULN) and Non-responders (HBV DNA ≥0.7MEq/mL) discontin-ued therapy at Week 48.Virologic Responders (HBV DNA <0.7MEq/mL but ALT ≥ 1.25 × ULN) continued therapy into the second year,and received blinded treatment up to Week 96. Virologic responderswho became Responders or Non-responders during the second yearof dosing discontinued therapy.Results Overall, 286/325 (88%) of ETV patients and 253/313(81%) of LVD achieved Response through Week 96. By Week 96, thecumulative proportion of patients who achieved confirmed HBVDNA <300 copies/mL by PCR was significantly higher (p < 0.0001)for the ETV- (94%) than for the LVD-treated group (77%). Thecumulative proportion of patients who achieved confirmed ALT nor-malization (≤1.0 × ULN) was higher for the ETV-treated group(89%) than for the LVD-treated group (84%). The safety profile ofETV was comparable to LVD.Conclusions Continuing through year-2, ETV was superior toLVD in achieving undetectable HBVDNA (<300 copies/mL).The fre-quency of achieving a Response was higher in the ETV- than in theLVD-treated group. The safety profile of ETV was comparable toLVD through year-2.

#179

Telbivudine preclinical safety studies suggest minimalrisk of chronic toxicity, reproductive toxicity orcarcinogenicityEDWARD BRIDGES1

1Idenix Pharmaceuticals, Cambridge, MA

Background A series of preclinical studies were undertaken tosupport clinical development of Telbivudine, a nucleoside for thetreatment of chronic hepatitis B, including chronic toxicity, carcino-genic potential, and reproductive toxicity. In these studies, telbivu-dine was investigated in large multiples of the human clinicalmaximum plasma concentrations (Cmax) and total exposure (areaunder the curve, AUC) achieved at the recommended human dose,600mg/day.Methods & Results Chronic toxicologic studies conducted over 6months in rats and 9 months in cynomolgus monkeys displayed notelbivudine-related effects on physiology. The no-observed-adverse-effect level for chronic dosing was considered to be 1000mg/kg/day

(10 × Cmax, 6–8xAUC). In a 2-year carcinogenicity study, there wasno evidence of oncogenicity in Sprague-Dawley rats treated with tel-bivudine at dosages up to 2000mg/kg/day (15 × Cmax, 14 × AUC) for≥85 weeks. Carcinogenicity was also evaluated in CB6F1-TgrasH2transgenic-mice. After 26 weeks, no differences were found in the incidence of neoplastic or non-neoplastic changes, comparing thehigh-dose (2000mg/kg; 31 × Cmax, 14 × AUC) group and control.Developmental and reproductive toxicity displayed no significanteffects in general reproductive fertility in rats treated with 2000mg/kg/day (15 × Cmax, 14 × AUC) and no effects on embryo-fetal or post-natal development in rats and rabbits treated with 1000mg/kg/day(10–19 × Cmax, 6–37 × AUC).Conclusions Large multiples of anticipated telbivudine exposurein humans were evaluated for chronic toxicity, carcinogenicity, andreproductive and developmental toxicity and shown to have no sig-nificant toxic effects in relevant animal models, suggesting minimalrisk for cumulative toxicity, carcinogenicity, or reproductive toxicityin humans.

#180

A phase III comparative trial of telbivudine andlamivudine for treatment of chronic hepatitis B inChinese patients: first-year resultsJIN-LIN HOU,1 YOU-KUAN YIN,2 DAO-ZHEN XU,3

D TAN,4 JUNGI NIU,5 XIA-QIU ZHOU,6

YUMIN WANG,7 LIMIN ZHU,8 YONGWEN HE,9

H REN,10 MOBIN WIN,11 C CHEN,12

SHAN-MING WU,13 Y CHEN,14 JIA-ZHANG XU,15

Q WANG,16 L WEI,17 GEORGE CHAO,18 B FIELMAN,18

NATHANIEL BROWN,18 JI-DONG JIA19

1NanFang Hospital, First Medical University of the PLA,2Shanghai Hua Shan Hospital, 3Beijing Ditan Hospital, 4XiangYa Hospital, 5No.1 Hospital, Changchun, 6Ruijin Hospital,Shanghai Second Medical University, 7Xi Nan Hospital,Affiliated to No.3, 8Tianjin Infectious Diseases Hospital, 9Xie HeHospital, 10Chongqing Medical University 2nd AffiliatedHospital, 11Shanghai Changhai Hospital, 12Shanghai LiverDisease Research Center, 13Shanghai Infectious DiseasesHospital, 14The First Affilated Hospital, 1581st Hospital P.L.A.Nanjing, China, 16First Hospital of Peking University,17Peking University People’s Hospital, 18Idenix Pharmaceticals,19Beijing Friendship Hospital

Background In a large Phase III trial (GLOBE), telbivudinedemonstrated greater efficacy vs. lamivudine in adults with chronichepatitis B.The present trial was undertaken to obtain additional datain Chinese patients.Methods This randomized double-blind trial is comparing 2 yearsof treatment with telbivudine or lamivudine in Chinese adults withchronic hepatitis B. Key entry criteria: HBsAg-positive, HBV DNA

Responseat Week 52 Telbivudine Lamivudine P value

n 167 165log10 HBV DNA ↓ 622 5.40 0.0004% PCR regative 70 43 <0.0001% ALT normalization 89 76 0.0033% Therapeutic Response† 87 64 <0.0001% HBeAg loss‡ 31 20 0.0473% HBeAg seroconversion‡ 25 18 0.1369% Resistance 4.5 10.1 0.0542

†HBV DNA <5 log10 copies/mL with HBeAg loss or ALT normalization‡HBeAg-positive patients


>6 log10 copies/mL, ALT 1.3–10 × ULN, HBeAg positive or negative,compensated liver disease. Results at 1 year are presented.Results The intent-to-treat population comprises 332 patients(290 HBeAg positive; 42 HBeAg negative). At week 52, telbivudinedisplayed superior antiviral efficacy (HBV DNA reduction) comparedwith lamivudine. Telbivudine was also superior for HBV DNA clear-ance by PCR assay, ALT normalization, HBeAg loss, and Therapeu-tic Response. Both treatments were well tolerated.Conclusions Telbivudine exhibited superior antiviral and clinicalefficacy vs. lamivudine in Chinese patients after one year, and waswell tolerated.

#181

Single-dose and steady-state pharmacokinetics andsafety of telbivudine following oral administration inhealthy Chinese subjectsP HU,1 J JIANG,1 H WANG,1 K PIETROPAOLO,2

G CHAO,2 N BROWN,2 X ZHOU2

1Peking Union Medical College Hospital, Beijing, China, 2IdenixPharmaceuticals, Cambridge, MA, USA

Background Telbivudine is undergoing phase III clinical evalua-tion in patients with chronic hepatitis B (GLOBE trial). To supportthis program, the present study describes the pharmacokinetics of telbivudine in healthy Chinese subjects.Methods Healthy Chinese subjects received either a single-dose of200mg, 400mg or 800mg (10 subjects/dose), or multiple-doses of600mg/day (12 subjects) of telbivudine. Single-dose (D1) and steady-state (SS) pharmacokinetic results are reported and compared to his-torical data obtained from non-Chinese subjects.Results Telbivudine was rapidly absorbed and exhibited dose-related plasma exposure. Principal D1 and SS pharmacokinetic para-meters in healthy Chinese subjects are comparable to previous studiesconducted in healthy non-Chinese subjects (Table). Telbivudine waswell tolerated, with no SAEs. The nature and frequency of AEs werecomparable to those reported previously.Conclusions Telbivudine was well tolerated in healthy Chinesesubjects with an overall safety and PK profile similar to that observedin non-Chinese subjects.

PK parameters from 600 mg/mL dose group

AUCτ (μg/mL · h) Cmax(μg/mL) Tmax(h) T1/2(h) CLR*

D1 SS D1 SS D1

Chinese 21.9 ± 7.7 26.1 ± 7.2 3.7 ± 1.2 3.7 ± 1.2 2.0 (0.5 − 3.0) 39.4 ± 12.1 6.5 ± 1.5Other 18.3 − 27.2 16.5 − 29.0 2.7 − 3.7 2.2 − 3.7 2.0 − 4.0 41.1 − 43.2 7.6 − 10.3

*CLR, renal clearanceResults from the Chinese study are presented as mean ± SD (median and range for Tmax). Study mean ranges are shown for results from other studies

#182

Telbivudine (LdT) compared to lamivudine fortreatment of chronic hepatitis B: results fromGLOBE, an international phase III trialCHING-LUNG LAI,1 EDWARD GANE,2

SATAWAT THONGSAWAT,3 YUMIN WANG,4

JENNIFER HEATHCOTE,5

YOUNG-MYOUNG MOON,6 YAGANG CHEN,7

WEI-LUN LU,8 JUNGI NIU,9 SENG-GEE LIM,10

JIN-LIN HOU,11 YOU-KUAN YIN,12

NIKOLAI NAOUMOV,13 NATALIE BZOWEJ,14

GEORGE CHAO,15 BARBARA FIELMAN,15

NATHANIEL BROWN,15

THE GLOBE STUDY GROUP15

1University of Hong Kong, 2Middlemore Hospital, Auckland,NZ, 3Chiang Mai University, Chiang Mai,Thailand, 4MilitaryMedical University, Chongqing, China, 5Toronto WesternHospital,Toronto, ON, Canada, 6Severance Hospital, Seoul,Korea, 7Zhejiang University College of Medicine, Hangzhou,China, 8No. 3 Hospital Zhong Shan Medical University,Guangzhou, China, 9Jilin University 1st Hospital, Changchun,China, 10National University Hospital, Singapore, 11NanFang

Hospital, No. 1 Military Medical University, Guangzhou,China, 12Huashan Hospital, Medical Center of FudanUniversity, Shanghai, China, 13University College, London, UK,14Sutter Health, San Francisco, CA, USA, 15IdenixPharmaceuticals, Cambridge, MA, USA

Background and Methods The GLOBE study is a randomizedblinded 2-yr trial comparing telbivudine (600mg/d PO) vs standardlamivudine (100mg/d PO) in 1,367 adults with chronic hepatitis B.Key entry criteria were HBsAg+, HBV DNA >6 log10 copies/mL byCOBAS PCR assay, ALT 1.3-10 xULN, and compensated liverdisease. Results of the primary analysis is at Week 52 are presented.Results Telbivudine was superior to lamivudine on all direct mea-sures of antiviral efficacy at Week 52. Telbivudine was superior tolamivudine in HBeAg+ patients for Therapeutic Response and His-tologic Response, and similar to lamivudine for these endpoints inHBeAg− patients. Available Week 76 data suggest that responsesincrease further during the second year. Both drugs were well-tolerated with similar adverse-event profiles.Conclusions After 1 year, telbivudine exhibited superior antiviralefficacy compared with lamivudine in HBeAg-positive and HBeAg-negative patients, with significantly less treatment failure and resistance.


#183

Functional analysis of HNF3 binding sites of HBVgenome in the inhibition effect of HNF3 on HBV replicationHONG TANG,1 FEI-JUN HUANG,2 FANG HE,1

LI LIU,1 SHU WANG,1 FENG-JUN LIU,1

TAO-YOU ZHOU,1 LIAN-SAN ZHAO1

1Center Of Infectious Diseases,West China Hospital of SichuanUniversity, Chengdu 610041, P.R. China, 2Department ofForensic Pathology, Medical School of Basic and ForensicSciences, Sichuan University

Background and Objective We showed previously that hepato-cyte nuclear factor 3 (HNF3) inhibits HBV replication. The aim ofthis study is to investigate the functional domain and molecular mech-anism(s) involved in the HNF3 inhibition of HBV replication.Methods The functional domain of HNF3β responsible for regu-lating of HBV replication was examined in NIH3T3 cells using areplication-competent HBV construct pHBV4.1 and a series of trun-cated HNF3β expression plasmids. By mutagenesis method, a set ofpHBV4.1HNF3mut constructs with mutations at the individualHNF3 binding sites in the HBV genome have been made.The impor-tance of each individual HNF3 site in the inhibition role of HNF3on HBV replication has been investigated.Results We show here that the N-terminal transcriptional activa-tion domain of HNF3β is primarily responsible for inhibiting viralreplication whereas the C-terminal transcriptional activation domainof HNF3β did not greatly affect HBV DNA synthesis. Mutations ofthe seven HNF3 binding sites of the HBV genome (XpHNF3 site1&2, CpHNF3 site1&2, PpHNF3, PS1pHNF3 and SpHNF3 site)have been shown to inhibit HNF3 from binding to the HBV HNF3sites by gel-shift assay. Analyze using these pHBV4.1HNF3mut con-structs have indicated that mutation of each HNF3 site in the HBVpromoters does not obviously affect the inhibition effect of HNF3.Conclusions This study demonstrated that the N-terminal tran-scriptional activation domain of HNF3β is important for the inhibi-tion role of HNF3, and inhibition of HBV replication by HNF3 isnot mediated through a single HNF3 site in the HBV genome.

#184

Establishment of a rapid and convenient HBVreplication mouse model by a hydrodynamic-based in vivo transfection procedureFENG-JUN LIU,1 LIN-YU DENG,2 HONG TANG,1

FANG HE,1 TAO-YOU ZHOU,1 SHU WANG,1

LIAN-SAN ZHAO1

1Center of Infectious Diseases,West China Hospital of SichuanUniversity, Chengdu 610041, P.R. China, 2School of LifeScience, Sichuan University, Chengdu 610041, P.R. China

Background and Objective Understanding the mechanism ofHBV replication may provide potential targets for developing anti-HBV treatment. To establish a small and convenient animal modelwith highly HBV replication, can be used for functional studies ofHBV.Methods HBV replication competent plasmid was transferred intoBALB/C mice via tail vein by hydrodynamic-based in vivo transfec-tion procedure. Mice were sacrificed at different time after injectionand collected the livers. Liver HBV DNA replication intermediate wasanalyzed by Southern Blot and HBcAg was checked by immunohis-tochemical technique; Serum HBsAg and HBeAg were detected byELISA.Results After hydrodynamic-based transfer of the HBV replicationcompetent construct, high level of HBV DNA replication intermedi-ates and HBcAg were detected in the mouse liver and HBsAg &HBeAg were detected in the serum, suggesting that HBV was repli-cated in the liver and secreted to the serum of the mouse. HBV DNAreplication intermediates were detectable on day 1 and abundant onday 3, the levels were slightly decreased and stay at a relative stablelevel between days 4 and 7, and was almost undectable on day 10after in vivo transfection. The expression patterns of HBV-specificantigens in the liver and serum were similar to that of HBV DNA. Inaddition, no obvious differences of HBV DNA replication interme-diates were observed in different parts of the mouse liver.Conclusion In this study, a rapid and convenient mouse modelwith high level HBV replication and expression was developed, whichprovided a very useful tool for the functional study of HBV.

Response HBeAg positive HBeAg negative

Telbivudine Lamivudine Telbivudine Lamivudine

Wk 52 Wk 76 Wk 52 Wk 76 Wk 52 Wk 76 Wk 52 Wk 76

n 458 163 463 165 222 68 224 67log10 HBV DNA ↓ 6.5* 6.6* 5.5 5.2 5.2* 5.3 4.4 4.7% PCR regative 60* 69* 40 41 88* 84* 71 67% Histologic Response 65* — 56 — 67 — 66 —% ALT normalization 77* 78* 75 68 74 76 79 64% Therapeutic Response† 75* 75* 67 58 75 75 77 70% HBeAg loss 26 40* 23 26 — — — —% HBV resistance 3* — 8 — 2* — 9 —% 1° Treatment Failure‡ 5* — 13 — <1 — 3 —

†HBV DNA <5 log10 copies/mL with HBeAg loss or ALT normalization‡HBV DNA never <5 log10 copies/mL*p < 0.05, telbivudine vs lamivudine


#185

Investigation of hepatitis B viral insertions in humanhepatocellular carcinomaNATHALIE WONG,1 WAH NG,1 DAVID LAM,1

HENRY CHAN,2 DAVID SMITH,3 PAUL LAI4

1Dept of Anatomical and Cellular Pathology,The ChineseUniversity of Hong Kong, 2Dept of Medicine and Therapeutics,The Chinese University of Hong Kong, 3Laboratory of Medicine& Pathology, Mayo Clinic Cancer Centre, Rochester, USA,4Dept of Surgery,The Chinese University of Hong Kong

Chronic infection of the viral hepatitis B (HBV) is a major etiologicrisk factor in the development of hepatocellular carcinoma (HCC).Despite viral DNA integration into the tumor genome is a commonphenomenon, the role of HBV integrants in liver carcinogenesisremains unclear. In an effort to gain insights into the viral integrants,we examined 15 HCC cell lines and 30 primary tumors that werederived from chronic carriers of HBV by the high-throughput analy-sis of restriction site-PCR. Interruption of cellular genes was indi-cated at chromosome locations including 2q14.2, 2q35, 6p11.1,8p11.21, 16q21 and 19q13.12. Alignment of integrants suggested thesites of viral insertion to occur in common chromosome fragile sitesand within the vicinity of important cancer-related genes, includingcadherin-11 (CDH11), astrocyte-derived trophic factor 1 (ATF1),ankyrin (ANK1) and transformation/transcription domain-associatedprotein (TRRAP). Of particular interest was consistent insertion ofviral sequences being found in introns or non-coding regions of thegenome. Disruption of viral sequences, on the other hand, involvedbreakpoints within a 500bp sequence between the core and HBxregions. In summary, viral integrants juxtaposition to cellular genesmay have implication in a disrupted gene function that may confergrowth advantages to infected hepatocytes. Consistent with the notionthat HBx is a transactivator of host cellular genes, recurrent truncatedHBx sequences observed in this study provide further support for itsrole in HBV-induced liver carcinogenesis.

#186

Association between chronic hepatitis C and thyroidautoimmunity and dysfunction in patients withhaemophiliaRAIMONDS SIMANIS,1 AGNIS ZVAIGZNE,2

LUDMILA VIKSNA,3 SANDRA LEJNIECE,4

AIVARS LEJNIEKS5

1Latvian Hepatologists Association, 2American Association ofClinical Endocrinologists, 3The European Association for theStudy of the Liver, 4European Haematology Society, 5AmericanDiabetes Association

Introduction Autoimmune thyroid diseases are complex condi-tions caused by an interaction between genetic and environmentalfactors. One environmental factor that could trigger autoimmunethyroid diseases in susceptible individuals is chronic hepatitis C. Somestudies has showed link between those two, however it is not knowwhether it is affected by different hepatitis C virus genotypes.Materials and Methods We investigated thyroid autoimmunityand function in 35 patients with haemophilia and chronic hepatitis Cand 40 control subjects by measuring free thyroxin (FT4), thyroidstimulating hormone (TSH), thyroid peroxidase antibodies (TPO)and antibodies against TSH receptors.Results We found no statistically significant difference in preva-lence of thyroid autoimmunity and dysfunction between two groups.However, there was statistically higher titer of anti TSH receptor anti-bodies in chronic hepatitis C group compared to healthy volunteergroup. Further analysis of relationship between different hepatitis Cvirus genotypes and level of thyroid hormones, we found FT4 to be

significantly higher and TSH significantly lower in patients with geno-type 2 compared to patients with genotype 1b virus (respectively FT417.38; 14.00pmol/l and TSH 0.92; 1.39 μIU/l).Conclusions Prevalence of thyroid autoimmunity and dysfunctionamong chronic hepatitis C patients is similar to the prevalenceobserved in healthy men. However, patients with chronic hepatitis Care at greater risk to develop clinically manifest thyroid disorder dueto significantly higher titer of antibodies against TSH receptors.Patients with hepatitis C virus genotype 2 are more susceptible todevelop thyroid dysfunction.

#187

Hepatitis C virus enzyme immuno assay, help toapproved the blood saftey or waste the blood supply?MOHAMMAD ALI JALALI FAR,1,2

JAMAL TORABI ZADEH,1,2 MOSTFA PARIDAR,1,2

MAJID MIAH ARAB,1,2 SHOHREH SAATI,1,2

SEYD MEHDI SAJADI,2 MAHNAZ BABAAHMADI,3

AHMAD NASIMIAN4

1Khuzestan Blood Transfusion Service, 2Iranian BloodTransfusion Organization Center, 3Sanaie Folad, 4Jondi ShapourMedical University

Blood screening with high sensitive methods is major factor ofenhance blood safety; reach to zero risk point of blood transfusion,establishment of physician confident and insurance of their patients.For enhance blood safety any positive history blood donor’s perma-nent deferred. At now the hepatitis screening tests reached tomaximum of their sensitivity. This high sensitivity must be accompa-nied by high specification so that enhancement blood safety and avoidwasting the blood supply.In this descriptive survey we studied 19238 blood donors that referredto Ahwaz blood transfusion service from 21 April–3 October 2005.17973 male, 1265 female; 4178 single, 15057 married; 17–65 yearsold, mean33 years. We used Avicenna HCV test version 3 for screen-ing test and RIBA for confirmatory the positive EIA samples.We obtained 5.3% (1010/19238) reactive sample for HCV test.17.8% (179/1005) of them had not repeated reactions in duplicateretest (technical errors) and 26.6% (267/1003) of cord samples werenegative.The RIBA test result of 834 samples was as the follow: 77%(642/834) negative, 7.3% (61/834) positive and 15.7% (131/834) indeterminant. We found significant difference in married man andRIBA test.Although this EIA kit has high sensitive but its non specific reactionscause a threaten to blood supply, high stress to blood donors and extracosts for blood donors and blood transfusion organization andconfuse the physician. We recommend that used an other kit as theparallel with this EIA kit.

#188

Increased prevalence of Type-II diabetes mellitus inhepatitis C virus infection in western IndiaDEEPAK AMARAPURKAR,1 NIKHIL PATEL1

1Bombay Hospital & Medical Research Centre

Background Prevalence of Type-II diabetes mellitus (DM) inIndian patients with hepatitis C infection (HCV) is unknown.Methods This 2-year prospective study was planned on consecu-tive 200 patients with HCV (age = 45.9 ± 9.8 years; male : female =1.3 :1). Presence and duration of DM, genotype and probable dura-tion of HCV and presence of steatosis and cirrhosis were noted. Forcomparison, 200 patients each of hepatitis B infection (HBV) [age =38.2 ± 8.2 years, male : female = 1.3 :1] and of functional boweldisease (FBD) [age = 41.4 ± 5.6 years, male : female = 1.2 :1] were


analyzed for presence of DM. Data were analyzed using chi-squaretest.Results Characteristics of patients with HCV were as follows:genotype distribution- genotype 3 in 58%, genotype 1 in 39% andgenotype 2 in 3% patients; probable duration of HCV = 12.81 ± 8.2years; steatosis in 55(27.5%) patients; and cirrhosis in 88(44%)patients. Of these, DM was present in 44(22.2%) patients with meanduration of DM = 6.13 ± 2.3 years. HCV preceded DM in 29 patientsby 10.8 ± 2.3 years. DM was present in 11(23.4%) patients with genotype 3 and in 6(19.3%) patients with genotype 1. In patients withDM, cirrhosis and steatosis were present in 28(63.6%) and20(45.4%) patients, respectively, as compared to 60(38.4%) and35(22.4%) patients without DM. In patients of HBV and FBD, DMwas seen in 24(12%) and 19(9.5%) patients, respectively.Conclusion There is increased prevalence of DM in patients withHCV. HCV precedes the development of DM by a decade.There wasno difference in prevalence of DM in different genotypes. Cirrhosisand steatosis were more prevalent in HCV patients with DM thanwithout DM.

#189

Different genotypes behave differently in hepatitis Cvirus infectionDEEPAK AMARAPURKAR,1 NIKHIL PATEL,1

PRIYAMVADA RANE1

Background Natural history of hepatitis C infection (HCV) dueto genotype 3, which is predominant in India, is not well-studied.Thisstudy was planned on patients with HCV to compare natural historyof genotype 3 and genotype 1.Methods This 10-year prospective long-term follow-up study{mean follow-up period = 3.6 ± 1.4 (range = 1–10) years} was carriedout on 108 patients with HCV. Group-A comprised of 65 patients ofHCV with genotype 3 (mean age = 46.1 ± 11.3 years, male : female =1.09 :1) and group-B comprised of 43 patients of HCV with geno-type 1 (mean age = 44.2 ± 8.2 years, male : female = 2.3 :1). Demo-graphic features, clinical presentation and course, response totreatment (either Interferon-Ribavirin or Peginterferon-Ribavirincombination) and complications were noted in all the patients. Datawere analyzed using chi-square test and Student t test.Results Group-A (genotype 3) patients had significantly longerduration of infection before presentation (mean duration = 12.5 ± 5.2years) as compared to group-B (genotype 1) (mean duration = 8.6 ±5.3 years). Group-A patients had more fatty liver on histology(32.3%, 21/65 patients) as compared to group-B (18.6%, 8/43patients). Overall, sustained treatment response was significantlybetter in patients of group-A (87.5%, 21/24 treated patients) as com-pared to group-B (56.2%, 9/16 treated patients). There was statisti-cally no significant difference in mode of acquiring HCV, presence ofdiabetes, obesity or alcoholism, clinical presentation, stage of liverdisease, complications like decompensation or hepatocellular carci-noma and mortality.Conclusion HCV with genotype 3 was associated with longer dura-tion of clinically silent infection, higher occurrence of fatty liver onhistology and higher treatment response.

#190

Recombinant adenoviral vector expressing non-structure protein 3 of hepatitis C virus efficientlyinduce specific humoral and cellular immuneresponse in miceTAOYOU ZHOU,1 LIANSAN ZHAO,1 WEIHUA LI,1

HONG TANG,1 MIN CHEN,1 LI LIU,1 FANG HE1

1Westchina Hospital

Background/Objectives To evaluate the specific humoral and cel-lular immune response induced by replication-deficient recombinantadenovirus expressing non-structure protein 3(NS3) of hepatitis Cvirus (HCV) in miceMethods We constructed recombinant adenovirus plasmid(pAdEasy-GFP-NS3) in E.coli BJ5183 by co-transfecting the back-bone pAdEasy-1 with shuttle plasmid pAdTrack-NS3, and generatedreplication-deficient recombinant adenovirus RAd-NS3 expressingnon-structure protein 3 of Hepatitis C Virus in 293 cells. 108 pfu ofRAd-NS3 were inoculated intraperitoneally into BALB/c mice,while control group were injected with recombinant plasmid pRc-NS3 100 μg. All mice were boosted 3, 5 and 7 weeks later. Then theserum were collected to detect anti-HCV by ELISA and NS3 antigenspecific Cytotoxic T lymphocyte (CTL) activity in vitro were detectedby a standard 4-hr 51 Cr release assays.Results The data showed recombinant adenovirus RAd-NS3 couldinduce both humoral and cellular immune response. The anti-HCVpositive rate in sera samples of the mice inoculated with RAd-NS3were 100% with the peak titer 1 : 10000 and average antibody levelremains 1 :1000 within 10 weeks; meanwhile specific CTL activityreached 53.22%. There was significant difference between the testgroup and control group (P < 0.05). No obvious side effect wasobserved after injection in both groups.Conclusions The recombinant adenovirus could induce strongerspecific humoral and celluler immune response and might be as a candidate vaccine used for both prevention and therapy of HCV infection.

#191

The study of immunological properties of the humandendritic cells pulsed with HBV antigen in vitroYUAN LIU,1 JUN LI,1 YA-PING HAN,1

ZU-HU HUANG1

1Department of Infectious Diseases,The First Affiliated Hospitalof Nanjing Medical University

Aim We aim to observe changes in DCs maturation after HBVantigen loading and evaluate their efficacy.Methods Monocyte derived (MD)-DCs were cultured with HBsAgor HBcAg to produce HBV antigen pulsed DCs (characterized byimmunohistochemistry). DCs function was assessed in an autologousmixed leucocyte reaction (AMLR). The expression of cell surfacemolecules and the production of intracellular cytokines were detectedby four-color flow cytometry. Results: After loaded with HBsAg orHBcAg, DCs are not impaired in the function of activation and mat-uration (p > 0.05).The HBsAg-pulsed DC induced T cells to producemore IFN-γ than the other groups from the HBsAb-positive healthysubjects (previously vaccinated) (p < 0.05).This may due to the gen-eration of specific immune responses. The results come from threegroups of chronic hepatitis B (CHB) patients are as follows: 1) thepercentage of IFN-γ-positive CD8 T cells (Tc1) was greater inHBcAg-pulsed DC induced group than HBsAg-pulsed DC inducedgroup and unpulsed DC induced group (p < 0.05); 2)the percentageof IFN-γ-positive CD4 T cells(Th1) was greater in HBcAg-pulsedDC induced group and HBsAg-pulsed DC induced group thanunpulsed DC induced group. Conclusion: These results may beclosely related to the immune deficiency of the persistent HBV infec-tion.The level of HBcAg-pulsed DCs of CHB patients may still havea sufficient capacity to present antigen to autologous T cells and also,to induce Th1 and Tc1. These findings suggest that HBV antigen-pulsed DCs, especially HBcAg-pulsed DCs could be a suitable DC-based vaccine.


#192

Distribution and functional significance of lymphocytesubsets in the liver tissue of chronic hepatitis B patientsJUN LI,1 YA-PING HAN,1 YUAN LIU,1 NIAN CHEN,1

DONG LI,1 ZU-HU HUANG1

Aim To define the characteristic distribution and the role of lym-phocyte subsets in the liver tissue of chronic hepatitis B (CHB)patients.Methods The liver tissue was obtained by ultrasound guided liverbiopsy.The cell suspension was prepared by the method of using med-imachine from liver biopsy specimens.The cell surface molecules weredetermined using flow cytometry.Results (1) In the liver tissue of CHB patients, the proportion ofCD3+ CD4+ cells (Th cell) was significantly lower than in peripheralblood; and the proportion of CD3+ CD8+ cells (Tc cell) was signifi-cantly higher than in peripheral blood. (2) In the liver tissue of CHBpatients with low hepatitis B virus (HBV) DNA lever (HBV DNA <103 copies/ml), the proportion of Th cells and the ratio of Th/Tc weresignificantly lower than in CHB patients with high HBV DNA lever(HBV DNA > 106 copies/ml). (3) As the lever of ALT elevated inCHB patients, the proportion of CD56+ cell (NK cells) in the livertissue was significantly higher than in peripheral blood in the CHBpatients.Conclusion Our data suggested that the disequilibrium of specificand nonspecific cell immune was present in CHB patients, and thecharacteristic distribution of lymphocyte subsets is different betweenliver and peripheral blood.This difference may play an important rolein inflammation events of hepatic diseases and be associated with thelevels of serum HBV DNA.

#193

Treating chronic hepatitis C virus infection inindividuals on methadone maintenance: feasible and effectiveJOHN FARLEY,1,2 GAVIN WALTERS,1 WENDY SHUM,1

ANGELA FARLEY1

1Dr. John Farley Inc., 2University of British Columbia

Background HCV infections in Canada are primarily IVDU-related. Current treatment guidelines recommending the pegylatedinterferons and ribivarin combination regimens, approved in Canadawere registration-trials based, and target the mainstream population;very little data supporting treating HCV in IDUs exist.Objective We evaluate outcomes of those receiving current HCVstandard-o-f care (Peg IFN & Ribavirin) and concurrently onmethadone maintenance treatment (MMT), in a community (and /orcorrectional institution) “real-life” healthcare setting, with those notreceiving methadone.Methods 184 individuals began treatment between February 2003and April 2005. We report on the weeks 12 ([EVR] for GI, 4, 6) and24 ([EOT] for G2, 3) HCV RNA results as at January 10, 2006.Results 77 received MMT; 107 did not (Non-MMT). Genotypedistribution was similar for both groups; treatment completion ratesas at week 24 were similar. 21/30 and 35/of 50 (70%) were HCV RNAundetectable at week 12 in the MMT and Non-MMT G1 groupsrespectively. At week 24 this was 12/12 (100%) and 21/ 23 (91%)respectively for G2, 3.Conclusion The overall EVR response for G1 of 70% and EOTfor G2, 3 of 96% in this preliminary observational dataset suggestthat the outcome in both groups in a “real-life” setting compare quitefavorably to registration trials regardless of MMT. More effort shouldbe made to reach and treat this very important marginalized group.

#194

Anti-HBx in sera is a potential marker ofdevelopment from hepatitis B (HB) to liver cirrhosis(LC) and hepatocellular carcinoma (HCC)XIAODONG ZHANG,1 HANG ZHANG,1

LIANYING WU,1 LI SHA,1 SHUYE LIU,2

XINHUA SHAO,2 LIHONG YE1

1Institute for Molecular Biology, College of Life of Sciences,Nankai University, 2Department of Clinical Inspection,TianjinThird Central Hospital

Hepatitis B virus X protein plays an important role in the develop-ment of hepatoma. In order to demonstrate the clinical significanceof hepatitis B x antigen (HBxAg) and antibody against HBxAg (anti-HBx) in sera of patients, we generated a polyclonal antibody of rabbitagainst HBxAg. HBsAg/anti-HBs, HBeAg/anti-HBe and anti-HBcwere examined in sera of patients suffered from HB (n = 173), LC(n = 106) and HCC (n = 61). In the present study, HBxAg and anti-HBx were examined by enzyme-linked immunosorbent assay(ELISA) in these sera. The results showed that the positive rates ofHBxAg/anti-HBx were 8.7/10.4% for HB, 17.9/40.6*% (p < 0.05, vsHB) for LC, and 9.8/34.4*% (p < 0.05, vs HB) for HCC, respectively.In all patients, the positive rates in anti-HBx-positive cases (n = 114)were 7.0% for anti-HBs+ and 28.9%** (p < 0.01, vs anti-HBs+) forHBsAg+/HBeAg+/anti-HBc+, respectively. Both HBxAg and anti-HBx were totally negative in the sera of normal healthy people (n =213) as negative control. It is suggested that anti-HBx-positive in serais a potential marker of development from HB to LC and HCC.

#195

Fulminant hepatic failure: aetiology, clinical courseand outcome; a report from developing countryMOBIN KHAN,1 SHAHINUL ALAM,1

KHORSHED ALAM,1 SALIMUR RAHMAN,1

NOORUDDIN AHMAD,1 FAZAL KARIM1

1Bangabandhu Sheikh Mujib Medical University. Dhaka.Bangladesh

Background/Objective There are scarcity of report about fulmi-nant hepatic failure (FHF) from developing countries. Most reportsare from the developed countries. Aetiological and host factors aredifferent in developed and developing countries. Our study evaluatedthe aetiology, clinical course and predictors of outcome of FHF.Methodology We prospectively analyzed 80 consecutive patients ofFHF admitted during the period of January 2002 to December 2004,in the Department of Hepatology of Bangabandhu Sheikh MujibMedical University.Results Patients were of aged (mean ± SD) 36.49 ± 16.38yrs. Mostof the patients (64.4%) were young, male : female was 2 :1 Com-monest (40%) cause was hepatitis E virus other causes were hepati-tis B virus (31.3%), Non A to E virus (10%), drugs (3%). Despitesupportive and intensive care overall mortality rate was 65%. Com-monest (81.4%) cause of death was cerebral edema other causes wererenal failure, infection, bleeding and electrolyte imbalance. Death ratewas more (77.8%) in female specially in pregnant and hepatitis Bvirus related patient than other causes. Appearence of ascites, prolongprothrombin time and longer icterus encephalopathy interval are sig-nificantly (p < .05) higher among the nonsurvivors. Serum bilirubinlevel and age has got no significant (p > .05) influence on mortality.All the patients expired within 3 days of hospitalisation.Conclusion Fulminant hepatic failure has a fatal outcome. Hepati-tis E virus is the commonest cause and cerebral edema is the com-monest and dangerous complication. Improvement of sanitation andhygienic condition and supply of safe drinking water for preventionof hepatitis E virus is strongly recommended.


#196

Progression of asymptomatic viremic carriers to E antigen negative chronic hepatitis B: a 3-yearlongitudinal studySANG HOON AHN,1,2 CHANG MO MOON,1

KI JUN SONG,3 DO YOUNG KIM,1 HWA SOOK KIM,1

JA KYUNG KIM,1 YONG HAN PAIK,1,2

KWAN SIK LEE,1,2 CHAE YOON CHON,1,2

KWANG-HYUB HAN,1,2 YOUNG MYOUNG MOON1,2

1Dept. of Internal Medicine, 2Institue of Gastroenterology, 3Dept.of Biostatistics,Yonsei University College of Medicine, Seoul,Korea

Background/Aims The long-term changes in patients with nega-tive e-antigen (HBeAg), especially in Asian patients, remain unclear.Moreover, so called “Asymptomatic viremic carrier (AVC)” referringto the positive HBsAg without HBeAg or aminotransferase elevationsand with HBV-DNA levels more than 100,000 copies/mL has no con-sensus definition and diagnostic criteria for clinical terms (Hepatol-ogy. 2004). To address these issues, we conducted a 3year-longitudinal study.Methods Between January 1999 and March 2004, a total of 157patients with HBeAg negative HBV infection without liver cirrhosisor hepatocellular carcinoma were regularly monitored every 3 monthswithout treatment. All patients were stratified into inactive carrier(IC), AVC, or chronic hepatitis (CH). Serum HBV-DNA was mea-sured by a hybridization assay (sensitivity: 1.4 × 100,000 genomes/ml). The statistical analysis of generalized estimating equation wasused for cumulative probability of changes.Results The median age was 42.7 years (M:F = 2.3 :1). By singletime-point observations, 3 year-cohort prevalence of HBeAg negativeCH varied between 12.7 and 35.8%. HBeAg-negative CH accumu-lated over time (p = 0.002) and transition rates among groups after3 years of follow-up are as follows: IC to CH, 6.0%; IC to AVC, 4.1%;AVC to CH, 23.2%. AVC seemed to be in the middle of transitionfrom IC to CH. A proportion of transition from IC to AVC or CHindicates that the hybridization assay method may be irrelevant to dif-ferentiate IC from CH.Conclusions We demonstrated the long-term changing patterns ofclinical status in HBeAg-negative HBV infection and clinical rele-vance of AVC state.

#197

Inhibition of HBsAg secretion by adenovirus-mediated RNA interference in vitro and in vivoYANG ZHENG GANG,1 JIN HAN YING,1

SUN DAN NA,1 CHEN ZHI*1

1Institute of Infectious Diseases, First Affiliated Hospital, Schoolof Medicine, Zhejiang University

To investigate the role of adenovirus-mediated RNA interference inthe expression of HBV surface antigen (HBsAg) in vitro and in vivo.Methods an U6 promoter cassette and an RNAi sequence activeagainst HBsAg were acquired by PCR method and cloned into thepShuttle vector. Recombinant adenoviral plasmids are selected onkanamycin and confirmed by restriction digest.The recombinant ade-noviral plasmid is transfected into Hek-293 cells. The recombinantadenovirus was harvested from Hek293 cells and transferred intoHepG2.2.15 cells, a cell line stably expressing HBsAg, in vitro andinjected into a HBV transgenic murine model in vivo using a hydro-dynamic injection method. The expression of HBsAg was measuredby radioimmunoassay.Results We successfully constructed a recombinant adenovirusvector which can express small hairpin RNAs targeting HBV surfacesequence site from 579 to 597. High doses of recombinant adenovirus

used for mice intravenous injection was acquired. A reduction ofHBsAg by approximately 61%, compared with empty-vector controls,was detected in HepG2.2.15 cells and a reduction about 47% in HBVtransgenic mice.Conclusion Adenovirus-mediated RNA interference can suppressHBsAg secretion in vitro and in vivo. The ground work of anti-HBVtherapeutic potential for the use of adenovirus vectors in vivo wasestablished.

#198

Recombinant interferon-beta-1a plus ribavirin in thetreatment of chronic HCV infection: a prospective,randomised, comparative pilot studySANG HOON AHN,1,2 YONG SOO KIM,1

HYUN WOONG LEE,1 JA KYUNG KIM,1

DO YOUNG KIM,1 YONG HAN PAIK,1,2

KWAN SIK LEE,1,2 KWANG-HYUB HAN,1,2

CHAE YOON CHON,1,2 YOUNG MYOUNG MOON1,2

1Dept. of Internal Medicine, 2Institute of Gastroenterology,YonseiUniversity College of Medicine, Seoul, Korea

Despite the advancement in the treatments, some patients withhepatitis C virus (HCV) infection are unable to achieve sustainedvirological response (SVR). Interferon (IFN)-β has been shown tohave anti-viral activity, which may be of use in treating viral infec-tions. The current prospective, randomized, pilot study is the first tocompare the efficacy of recombinant IFN-β-1a (44mcg) given sub-cutaneously 3 times a week plus oral ribavirin (1000–1200mg/day)versus IFN-α (3MIU) plus ribavirin therapy.Methods A total of 20 treatment-naïve patients were randomizedequally.The duration of treatment was 24 weeks followed by 24 weeksof observation. Patients who did not achieve early virological response(defined as a decreased of at least 2 log viral load from baseline) attreatment week 12 were classified as non-responder and discontinuedfrom the treatment but continued with the observation phase.Results At end of treatment, 6 patients (60%) and 4 patients (40%)in the IFN-β-1a group and IFN-α group respectively achieved viralclearance. The SVR at the end of the observation period was similarin both groups (40%). However, the baseline viral load in the IFN-β-1a group was significantly higher (p = 0.034) and the IFN-β-1agroup had more HCV genotype 1 patients. IFN-β-1a group appearedto have a better safety profile compared to IFN-α group.Conclusion IFN-β-1a combination attained a similar SVR rate asIFN-α combination. The former combination was associated withmilder adverse events in term frequency and severity. A largerprospective study will be required to confirm the current findings.

#199

Pyrosequencing-based approach for rapid detection ofHepatitis B Virus infection resistant to lamivudine,adefovir and entecavirCHENGYAN MENG,1 JIALING JIN,1

WENHOGN ZHANG*1,1

1Huashan Hospital, Fudan University

Background The emergence of drug-resistant viral variants is theinevitable consequence of incomplete suppression of hepatitis B virus(HBV) replication during treatment with nucleotide analogues.Determining the resistance profiles of these variants has becomeincreasingly important in the clinical management of HBV infectionHere we have developed a pyrosequencing assay for the rapid char-acterization of resistance to all the current available nucleotide analogues.Method Pyrosequencing is a real-time DNA sequencing techniquegenerating short reads rapidly and inexpensively. We have developed


the method to detect a series drug resistance genotypes by this tech-nology. Four oligonucleotide probes covering 3 domains with thecapability to detect 8 genotypes of drug resistance were designed,including L180M, A181T, M204V/I/S for lamivudine resistance,A181T/V and N236T/N238D for Adefovir resistance, and S184Gand S202I for entecavir resistance. A small number of serum samplesfrom patients with phenotypic resistance to lamivudine and someHBV polymerase/reverse transcriptase (POL/RT) clones developedby site-directed mutagenesis, with mutations of A181T/V, N236T,N238D, S184G, and S202I, were used to evaluate the methid ofpyrosequencing.Results and Conclusion Comparing the direct Sanger sequenc-ing, the method of pyrosequencing could detect all the drug resis-tance related genotypes of HBV POL/RT. The rate of concordancebetween the results obtained with the pyrosequencing for the detec-tion of rtL180, rtA181, rtS184, rtM204, rt N236 and rtN238 andthe results obtained by sequencing was 100%. Thus, the methodpyrosequencing is a reliable, low-cost and high throughput tool forthe detection of antiviral-resistant HBV.

#200

Extended lamivudine therapy in patients with chronichepatitis B infectionDEEPAK AMARAPURKAR,1 NIKHIL PATEL,1

RESHMA TIPNIS1

1Bombay Hospital & Medical Research Centre

Background There are scanty data on extended Lamivudinemonotherapy (>1 year) for chronic hepatitis B infection (CHB) inIndian patients.Methods A long-term follow-up study of Lamivudine monother-apy (100mg/day PO) was planned in CHB (n = 382; age = 44.5 ±12.2 years; male : female = 5.06 :1; HBeAg-positive/HBeAg-negative= 192/190; chronic hepatitis/compensated cirrhosis/decompensatedcirrhosis = 226/53/103). Data regarding compliance, efficacy andresistance were assessed.Results Of 382 patients, analysis was performed on 242 patientswho completed at least 1-year treatment. In HBeAg-positive patients(n = 110), on-treatment seroconversion rate was 25.4% at 1 year, 31%at 2 years, 26.4% at 3 years, 21% at 4 years and 16.5% after 5 years.Sustained response rate was 69.2%, but further 33% experiencedrelapses. In HBeAg-negative patients (n = 132), on-treatment viro-logical response was seen in 60% at 1 year, 70.6% at 2 years, 43.2%at 3 years, 31.8% at 4 years and 25% after 5 years. Sustained responserate was 30.5%, but further 50% experienced relapses. Overall,Lamivudine resistance rate was 16.1% at 1 year, 31% at 2 years, 38%at 3 years, 45% at 4 years and 62% after 5 years. Of these, 20% pre-sented with clinical decompensation. With Adefovir in patients withLamivudine resistance, 70% achieved on-treatment response. Duringstudy, compliance was 86% and mortality was 6%.Conclusions Extended Lamivudine therapy improves responserates for initial 2 years. Emergence of resistance during therapy andfrequent relapses after stopping therapy are major problems. Adefovirleads to clinical stabilization in majority of patients with Lamivudineresistance.

#201

Determination of platelet-activating factor (PAF) by reversed phase high-performance liquidchromatographic technique (rHPLC) and itsapplication in viral hepatitisHONG-CUI CAO,1,2 XIAO-MING CHEN,1,2 WEI XU1,2

1Department of Infectious Disease, 1st Affiliated Hospital,Medical School, Zhejiang University, 2Key Laboratory ofInfectious Disease of Ministry of Health

Correspondence to Xiao-ming Chen, [email protected] To establish a new assay for platelet-activating factor(PAF), comparing with its bio-assay, and detect the plasma or serumlevels of tumor necrosis factor-α (TNF-α), malondialdehyde (MDA),endotoxin (ET) at the same time, and discuss their significance invarious types of viral hepatitis.Methods To measure PAF,TNF-α, MDA and ET levels in 60 con-trols, 16 cases with acute viral hepatitis, 71 cases with chronic viralhepatitis, 19 cases with severe viral hepatitis separately by reversedphase high-performance liquid chromatographic technique (rHPLC),bio-assay, ELISA, TBA and limulus lyssite test (LLT). Results TherHPLC is more convenient, sensitive, specific and less confusion, infine correlation comparing with bio-assay (r = 0.912, P < 0.01). Theplasma level of PAF in patients with viral hepatitis was higher thancontrols (P < 0.01), so did TNF-α, MDA and ET.Conclusion To detect PAF with rHPLC is more reliable and moreaccurate. The new assay has important significance in PAF research.It is indicated that the assay of plasma PAF level, combined withMDA, TNF-α and ET detecting, can be used to monitor the sever-ity of liver damage and push about the base and clinic researches ofvarious types of viral hepatitis.

#202

Construction and expression of EGFP controlled byHBV promoters in eukaryotic cellsXIAOYAN WANG,1 NA XIE,1 JUSHENG LIN1

1Institute of Liver Diseases,Tongji Hospital,Tongji MedicalCollege, Huazhong University of Science

Objective To construct four eukaryotic expression vectors con-taining the promoters of Pre-s gene, S gene, C gene and X gene ofhepatitis B virus, and detect green fluorescence protein expressioncontrolled by the promoters of HBV.Methods The P3.6II plasmid containing the entire HBV 3.5KbmRNA reverse transcriptional unit was used. The four promoterswere obtained by PCR amplification and then cloned into PGEM-Teasy vector. After double enzyme cutting, the promoter DNA frag-ments were inserted into eukaryotic expression plasmid pEGFP-1.The recombinant plasmid pEGFP-1/S promoter, pEGFP-1/C pro-moter, pEGFP-1/X promoter, pEGFP-1/Pre-s promoter were thentransfected into HepG2 cells using a routine lipofectamine method.The stable GFP expression was detected by fluorescence microscope.Results Double restriction enzyme digestion and DNA sequencingindicated successful construction of the eukaryotic expression vectors.The green fluorescence could be seen in transfected HepG2 cellsunder fluorescence microscope.Conclusion The recombinant expression vectors may lay the foun-dation for further studies whether or not four promoters of HBV arethe targets of the new antivirus drug.

#203

The deletion of La protein binding site leads toinstability of HBV RNA in HepG2YUAN-YUAN LIN,1 NA XIE,1 XIU-MIN ZHOU,1

YU-HU SONG,1 JU-SHENG LIN1

1Institute of Liver Diseases, Afffiliated Tongji Hospital of TongjiMedical College, Huazhong Univers

Objective To investigate the effect of the deletion of La proteinbinding site in HBV RNA on the stability of HBV RNA for furtherstudy on the significance of the site in HBV life cycle.Methods The HBV vectors with mutation which resulted in dele-tion of the La protein binding site in HBV RNA were constructedand then transfected into HepG2 cells, with the normal vectors as thecontrol. The HBV RNA secondary structure was calculated by com-


puter. The HBV RNA level was analyzed by semi-quantitative RT-PCR, and HBV antigens were tested by ELISA in each group.Results The mutant HBV vectors were successfully constructed.The HBV RNA secondary structure was disorganized after deletionof the site. The HBV RNA level and the expression efficacy of HBVantigens in the mutant group were significantly lower than those inthe control.Conclusion The sequence conservation of the site in HBV DNAis necessary for the formation of the special structure which is Laprotein binding site in HBV RNA, and the disorganization of thestructure due to mutation of the sequence in HBV DNA leads toinstability of HBV RNA in which the cleavage site in the upstream ofthe structure is exposed to endoribonuclease in human cells. The Laprotein binding site in HBV RNA plays a important role in HBV lifecycle.

#204

Viral specific T cells phenotypes and toll like receptorsmRNA expression in CD8 cells of chronic hepatitis BpatientsHAIYING ZHANG,1 CHEE-KIN HUI,1 NIKKI P LEE,1

YUI-HUNG YUENG1, JOHN M LUK1,GEORGE KK LAU1

Centre For The Study of Liver Diseases,The University of HongKong, Hong Kong SAR

Background The clearance or persistence of hepatitis B virus(HBV) is affected by HBV-specific cytotoxic T-lymphocyte (CTL).Since toll like receptors (TLRs) may contribute to the viral specificT cells. But what and how the TLRs take their functions in chronichepatitis B (CHB) patients remains unknown.Aims To investigate the association of TLRs in CD8+ T cells andtheir relationship with CTL cells in chronic hepatitis B patients.Material and methods Peripheral blood mononuclear cells(PBMC) were collected from 28 HLA-A2+ patients. Group A (n =8) were patients with spontaneous resolution of CHB (anti-HBc plusanti-HBs positive) while Group B (n = 20) was HBeAg (+) CHBpatients. Ex-vivo model:The PBMC of these patients were co-culturedwith live hepatitis B virus (HBV) for 7 days. PBMC were stained forthe frequency of CD4, CD8, CD45RA, CD45RO and CCR7 byFACS analysis. Viral specific CD8 cells were tested by tetramer toHLA-A2 limited HBV peptides. CD8+ cells were isolated and TLRs-2, 3 4 and 7 mRNA level in CD8+ cells and HBV DNA were testedby real time PCR. Interferon-g secretion was tested by ELISpot.Results After co-culture, there was an increase in HBV-specificCD8+tetramer+ cells in Group A when compared with Group B(mean ± SD 1.971 ± 0.694% vs. 0.7 ± 0.362%, respectively, P 0.001).Interferon-γ secretion was higher in Group A when compared with ingroup B on day 7 after stimulation (SFC were 304.25 ± 156.12 inGroup A and 49.6 ± 46.04 in Group B, P = 0.0002).The HBV DNAin the ex-vivo model was lower in Group A from day-5 to Day-7 ofco-culture. The frequency of CD8+CD45RA-CCR7-cells before co-culture was similar in both groups In both groups, 90% of the HBV-specific CD8+ cells were CD45RA-CCR7- effecter-memory T cells.TLR2, 3 and 4 mRNA were higher expressed in Group A than inGroup B. But only TLR4 has statistic significance (mean ± SD 2.915± 1.536 in Group A vs. 1.010 ± 0.988 in Group B respectively, p0.006).Conclusion HBV-specific CD8+tetramer+ cells are higher inpatients with spontaneous resolution of chronic HBV. TLR4 may beassociated with activation T memory cell in response to HBV.There were no significant differences among the phenotypes ofCD8/tetramer positive cells between the two groups.

#205

A fusion DNA vaccine that targets antigen-presentingcells enhanced the specific immune response toHbsAg in miceCHENG ZHOU,1 ZHI CHEN,1 GUOPING PENG1

1The Institute of Infectious Diseases, 1st Affiliated Hospital ofZhejiang University

Background DNA vaccine shows a great potential in anti-HBVimmunotherapy. However, most transfected cells are not the profes-sional antigen presenting cells (APC) which are critical for generat-ing the primary immune response. Cytotoxic T-lymphocyte antigen4(CTLA-4), a receptor expressed on activated T cells has a strongbinding affinity to B7 molecule, which are primarily present on APCs.Aim In this study, a DNA vaccine encoding a fusion protein ofHBsAg and murine CTLA4 was designed to explore its ability toenhance the HbsAg-specific immune response.Methods A DNA vaccine was constructed by fusing HBsAg geneand extramembrane domain of murine CTLA4 gene. The DNAvector was confirmed by restriction enzyme analysis, sequencing.After transfection, a fusion protein CTLA4-HbsAg expressed by theDNA vaccine was detected by Western blot and ELISA, and its B7binding activity was assayed by cytometry flow. Mice were immunizedwith DNA vaccine by intramusclur injection. The HbsAg-specificantibody and CTL were analyzed by ELISA and LDH-release assay.Results Immunization of mice with the DNA vaccine expressingthe fusion protein CTLA4-HbsAg was shown to induce acceleratedand increased Ab response and cytotoxic T lymphocyte responses ascompared with those receiving the HbsAg expressing vector.Conlusion These findings demonstrate that targeting HBV antigento APCs results in enhanced both humoral and cellular immuneresponse, which strongly indicates that this approach may be applic-able in improving both preventive and immunotherapy efficacy ofanti-HBV DNA vaccine.

#206

Study on the immune response against Hepatitis Bvirus induced by HBcAg recombinant adenovirus-transduced dendritic cells in BALB/c miceHONG-YU JIA,1 ZHI CHEN,1 YIN HUANG,1

LIN-FU ZHOU,1 WEI WU1

1Institute of Infectious Diseases, the First Affiliated Hospital,Medical College, Zhejiang Universit

Objective To study the immune function against Hepatitis B virusinduced by HBcAg recombinant adenovirus-transduced dendriticcells in vivo or vitro.Methods DCs were generated from bone marrow cells from theBALB/c mice, transfected with HBcAg recombinant adenovirus Ad-C (DC/Ad-C) or pulsed with HBcAg (DC/HBcAg) and used foranalysis of the phenotypic makers by flow cytometry and the stimu-latory capacity in MLR. HBcAg-specific activity of splenic CTL ofBALB/c mice was measured by LDH release assay and the anti-HBstiters in sera was examined by RIA after being immunized withDC/Ad-C, DC/HBcAg.Results Either the Ad-C-tranduced or HBcAg-pulsed DCsexpressed similar levels of phenotypic markers, and showed similarstimulatory capacity in either allogeneic or autologous MLR, respec-tively. DC/Ad-C more efficiently stimulated splenic Th and Tc tosecrete IFNγ than DC/HBcAg, and induced stronger splenic HBcAg-specific CTL responses than that induced by DC/HBcAg at 2 and 4weeks after immunization, respectively.Conclusion HBcAg recombinant adenovirus-transduced DCs caninduce Th1/Tc1 (type I) cell-mediated immunity and HBcAg-specificCTL, and maybe a efficient inducer in priming HBV-specific T cellresponse.


#207

Screening of thymosin alpha 1 binding protein byyeast two-hybrid systemHAIHONG ZHU,1 JIANZHONG CHEN,1

KEZHOU LIU,1 ZHI CHEN1

1Institute of infectious diseases, the first affiliated hospital,medical school, Zhejing University

Objective To screen Tα1 binding protein (TBP) interacting withTα1 from human T lymphocyte cDNA library.Methods The double stranded Tα1 gene was amplified by PCRusing chemical synthesized the signal strand Tα1 as template, and therecombinant pGBKT7-Tα1 was constructed according to the routinemethods. After verification by DNA sequencing, the pGBKT7-Tα1was transformed into yeast AH109, and the clones grown on SD/-Trp/X-α-Gal plate proved that the recombinant itself did not activateUAS downstream promoter to express downstream report gene.Thenthe recombinant plasmid and library plasmid were co-transformedinto AH109 by acetic acid lithium transforming method. Positiveclones grown after cultured at 30°C for 7 days. After identification ofpositive clones, the two clones were further sequenced to identify itsDNA sequence.Results After sequencing and alignment, the partial fragments ofthe two molecules are similar to actin-gamma and elongation factor-1 alpha, respectively.Conclusion Two molecules TBP1 and TBP2 that can interactingwith thymosin α1 were found by screening T lymphocyte cDNAlibrary using yeast two-hybrid system. The interactions of TBP1 andTBP2 with Tα1 were confirmed. The partial fragments of the twomolecules are similar to actin-gamma and elongation factor-1 alpha,respectively.

#208

Coinfection of HBV HCV in a cohort of HIV-infectedpaid blood donors from Henan province of ChinaWEIDONG ZHANG,1 DONGSHENG HU,1

GUANGCAI DUAN,1 TONG LI,2 HUI ZHUANG,2

FENGMIN LU2

1Department of Epidemiology, College of Public Health,Zhengzhou University, Henan, 450052, China, 2Department ofMicrobiology, Peking University Health Science Center, Beijing,100083, China

The commercial blood/plasma collection practices in the early 1990shave fueled the transmission of HIV in China. Hepatitis B and C viralinfections are highly prevalent among HIV-infected persons, becauseof shared transmission routes. However, limited data are available onthe epidemiology of this coinfection among paid blood donors inChina. HBV/HCV coinfection in a cohort of HIV-infected paid blooddonors from Henan province of China was studied. In a populationof 181 HIV seropositive blood donors, the coinfection rates of HIV+ HBV, HIV + HCV, HIV + HBV + HCV are 1.6% (3/181), 79%(140/181) and 2.2% (4/181) respectively. Significant high HIV/HCVcoinfection rate in HIV infected paid blood donors was same as otherparenteral transmission such as injection-drug use or transfusions.However, due to strict requirement of HBV screening to all blooddonors, the HBV/HIV coinfection rate was much lower than reported.Accumulating evidence indicated HIV might increase HCV viral loadand accelerate liver disease progression in persons with HIV/HCVcoinfection, together with the improved survival of AIDS patientsbenefited from HAART, have made HCV related chronic viral hepati-tis a major source of health burden. It is also worth to notice our datahere underlined that HIV/HCV co-infection increased the risk ofsexual transmission of HCV. It would also be a new challenge tocontrol the spread of HCV from individuals with HIV/HCV coinfec-tion to their sexual partners.

#209

Study on the immune response induced by HBcAggene-modified dendritic cells in Hepatitis B virustransgenic miceHONG-YU JIA,1 ZHI CHEN,1 YIN HUANG,1

LIN-FU ZHOU1

1Institute of Infectious Diseases, the First Affiliated Hospital,Medical College, Zhejiang Universit

Objective To investigate the immune response against HBVinduced by HBcAg recombinant adenovirus (Ad-C)-transduced den-dritic cells in HBV transgenic mice.Methods DCs were generated from bone marrow cells from theHBV-Tg mice, and transfected with Ad-C. HBV-Tg mice wasreceived DC/Ad-C twice at 3-week intervals. Intracellular cytokinesof splenic T cells, HBcAg-specific activity of splenic CTL and anti-HBc and HBV DNA titers in sera were detected after immunizationby flow cytometry, LDH release assay, ELISA and quantitative PCR,respectively.Results DC/Ad-C efficiently stimulated splenic Tc to secrete IFNγand induced stronge splenic HBcAg-specific CTL responses. Thesuppression of serum HBsAg and HBV DNA and the reduction ofexpression of HBcAg and HBsAg in liver in HBV-Tg mice inducedby DC/Ad-C were stronge.Conclusion HBcAg recombinant adenovirus-transduced DCs caninduce Tc1 and CTL response, and reduce the titer of serum HBsAgand HBV DNA, and reduce the expression of HBcAg and HBsAg inliver in HBV-Tg mice, and maybe a promising candidate for the ther-apeutic vaccine for chronic HBV infection.

#210

Molecular epidemiology of hepatitis B viral genotypesin Tibet: comparison with other regions of ChinaWENHONG ZHANG*,1 LINGYUN SHAO,1

XUEHONG GONG,2 HAILI YU,1 CHENGYAN MENG,1

JIALING JIN1

1Huashan hospital, Fudan University, 2Second People’s Hospitalof Tibet

Background In the countries like China where the population ismixed with people coming from many different ethnic origins, thegenotypes of hepatitis B virus (HBV) distribute geographically. Herewe compare the genotypes of HBV from patients of Tibetan nationin Tibet and patients of Han nation both from Tibet and Shanghai ofChina.Method The preS1/PreS2/S region of HBV genes from 146 chronichepatitis B (CHB) patients of Tibetan nation and 182 CHB patientsof Han nation from Tibet (58 cases) and Shanghai (124 cases) wereamplified by the polymerase chain reaction and sequenced. Phyloge-netic analysis of the nucleotide sequences was performed using neigh-bor joining or maximum parsimony methods of tree reconstruction.Results Tibetan HBV isolates were mainly classified in type D(86.7%, 131/146) and type C (9.6%, 14/146), with only one caselocated in the type B cluster. In comparison, HBV isolates from Hannation in Tibet are similar with those from Shanghai, predominantlywere classified as genotype C (83.9% in Shanghai and 81.0% inTibet) and genotype B (15.3% in Shanghai and 15.5%).Conclusion The genotypes of HBV not only distribute geograph-ically, but also can be kept in societies living closely together evenafter migration over long distances. Therefore, HBV genotypes mayadd additional support to anthropological data on ancient migrationevents.


#211

Detection of differentially methylated genes betweenone pair of identical twins with discordant phenotypeof chronic HBV infectionBAO-YAN XU,1 YU-MING WANG,1

GUO-HONG DENG,1 LIN LAN,1 YAN-PING HUANG,1

ZHAO-XIA TAN,1 SHI-TAO DING1

1Institute of Infectious Diseases of PLA, Southwest Hospital,Chongqing, China

Objective To analyse the differential methylation status of genomicCpG island DNA between one pair of identical twins (twin A andtwins B) with discordant phenotype of HBV infection and to discoverdifferentially methylated genes.Methods MCA coupled with RDA (methylated CpG island ampli-fication followed by representational difference analysis) methodswere applied. Briefly, genomic DNA was digested by isoschizomerswith distinct methylation sensitivity (Sma I/Xma I); Specific adaptorswere ligated to the digestion products; PCR was performed to enrichthe MCA amplicons; Representational difference analysis was per-formed using DNA of twin A as tester and DNA of twin B as driver.Then repeated once using DNA of twin B as tester and DNA of twinA as driver. After three competitive hybridizations, the differentialbands were isolated with the agarose gel electrophoresis analysis,cloned into T vectors and sequenced; Finally, sequencing results wereanalyzed by bioinformatics methods.Results Four genes with differential methylation status might con-tribute to the twins with discordant hepatitis B phenotype.They weregenes of OCA2 (oculocutaneous albinism II,15q11.2–q12), TJP3(tight junction protein 3,19p13.3), KIAA0543 (likely ortholog ofmouse SCO-spondin, 7q36.1) and LOC399839 (similar to doublehomeobox protein, 10q26.3) respectively. The former two werelocated in the intron regions of genome; KIAA0543 was located in

the extron region of genome; while LOC399839 gene is a novel genewith unknown function.Conclusion Differential methylation of genes occurs in identicaltwins with discordant phenotype of HBV infection. Whether thesechanges are involved in the pathogenesis of different phenotypesneeds further elucidation.

#212

Analysis of HBV Genotypes btween mnorities andHan patients with chronic hepatitis B in XinjiangYUEXIN ZHANG,1,2 LINA LI,1,2 HONG XU,1,2

XIAOBO LU,1,2 LIN XIAO,1,2 AMANGULI YASHENG1,2

1First Affiliated Hospital, 2Xinjiang Medical University

Objective To investigate the distributions of HBV genotypesamong different nationality chronic hepatitis B (CHB) patients inXinjiang.Methods The HBV genotypes among 6 nationality groups of CHBin Xinjiang were detected by genotypes speciality prime PCR. 410Sera samples come from Uygur (102), Han (226), Kazark (34), Hui(27), Mrgolia (13), Xibo (8) in different cities of Xinjiang.Results There were HBV genotype B (44.1%), genotypes B + Cmixture (32.9%), genotype C (19.0%), genotype D (1.2%), genotypeB + D mixture (1.7%) among the patients with CHB in Xinjiang(seeing Table 1). The 110 minorities patients with CHB had HBVgenotype B (59.8%), genotype B + C mixture (19.6%), Genotype C(12.5%), genotype B + D mixture (3.8%) and Genotype D (2.7%),226 Han patients with CHB had HBV genotype B + C (43.8%), geno-type B (31.4%) genotype C (24.3%) and no HBV genotype D.Conclusion Xinjiang Patients with CHB were mainly infected byHBV genotype B, C and D, but it is different HBV genotype infec-tion between Han patients and minority patients.

Table 1 Distribution of HBV genotypes between Han and minority patients with CHB

HBV genotype B C D B + C mix B + D mix Notyping Totaln % n % n % n % n % n % n

Han patients 71 (31.4) 55 (24.3) 0 (0) 99 (43.8) 0 (0) 1 (0.4) 226minoritiespatients 110 (59.8) 23 (12.5) 5 (2.7) 36 (19.6) 7 (3.8) 3 (1.6) 184Total 181 (44.1) 78 (19.0) 5 (1.2) 135 (32.9) 7 (1.7) 4 (0.9) 410

#213

Epidemiology of the hepatitis B and C viruses in HungaryJUDIT GERVAIN,1 HUNGARIAN VIRAL HEPATITISGROUP2

1Sub-Department of Hepato-Pancreatology, Szent GyörgyHospital, Székesfehérvár, Hungary, 2Members listed below

Background The different subtypes of the hepatitis B virus (HBVA–H) and the hepatitis C virus (HCV 1–6) have diverse pathogenic-ity and sensitivity to therapy. Therefore the HCV and HBV genotypedistributions have important clinical implications.Aim To reveal the genotype distribution of HCV and HBV in Hun-garian patients with chronic viral hepatitis in light of national data ontreatment outcomes.Methods In Hungary, genotype analysis of the serum samples ofpatients treated in the national hepatitis centres happen centralised inour laboratory. Samples are tested for HCV using a combination ofreverse transcriptase-polimerase chain reaction (PCR) and reversehybridization line probe assay, and for HBV using a combination ofnested-PCR and reverse hybridization line probe assay.

Results Between 1999–2005, genotype analysis was carried out for2054 HCV positive patients (male/female: 49%/51%). Between2003–2005, genotype analysis happened for 210 HBV positivepatients (male/female: 31%/69%).The genotype distribution of HCVwas 1a 4.7% (n = 97); 1b 86.8% (n = 1784); 1a + 1b 4.0% (n = 82);1a + 1b + 2 0.2% (n = 4); 1b + 2 1.0% (n = 20); 3 1.0% (n = 20); andmixed: 2.3% (n = 47).The genotype distribution of HBV was A 44.0%(n = 92); B 1.5% (n = 3); C 2.0% (n = 4); D 45.0% (n = 95); A + D4.7% (n = 10); and mixed 2.8% (n = 6).Conclusions 92% of the Hungarian patients with chronic viralhepatitis C are infected with the most resistant genotype HCV-1b,which may explain the lower rates of sustained responders to treat-ment in Hungary. Infections caused solely by HCV-2, -4, -5, and -6do not occur. The most common HBV genotypes are A and D.


#214

The correlation of genom mutations in HCV-1b withIFN-sensitivity in Hungarian patientsJUDIT GERVAIN,1 JUDIT SIMON,1 AGNES CZIBULA,2

TIBOR KALMÁR2

1Department of Public Health, University of Oxford, UK,2Biological Research Centre, Szeged, Hungary

Background Japanese researchers found that the structure of apart of the viral genom, namely the Interferon Sensitivity Determin-ing Region (ISDR: Aa 2209–2248), is associated with response tointerferon (IFN) therapy in chronic HCV-1b infection. Several inter-national studies could not confirm this correlation and showed thatthe virus quasispecies are different from HCV-J in Western Europe.Aims To analyse the virus quasispecies and to investigate the pre-dictive value of the ISDR mutations for IFN sensitivity in Hungary.Methods Pre-treatment samples of 21 patients, chronically infectedwith HCV-1b and treated with IFN-α, were analysed. Nested reverse-transcriptase polimerase chain reaction and direct sequencing wereused to investigate the nucleotide structure of the ISDR. Medianjoining network analysis was used to analyse the role of individualaminoacid substitutions.Results The structure analysis showed that the Hungarian quasi-species of HCV-1b differ from HCV-J.The prevalence of mutant typeISDR (≥3 aminoacid substitutions) was 0 out of 18 non-respondersand 2 out of 3 sustained responders. Six of the non-responders hada single arginin substitution at position 2218 in contrast to none ofthe sustained responders. Test of independence showed that IFN-sensitivity is highly associated with the structure of the ISDR (p = 0.004).Conclusions Despite the HCV-1b quasispecies different fromHCV-J in Hungary, our findings support that mutations in the ISDRof HCV-1b influence the outcome of IFN therapy. They suggest thatmutant type ISDR is predictive of sustained response and arginin sub-stitution in position 2218 is predictive of no response.

#215

Modulation of endoplasmic reticulum (ER) calciumsignalling affects Hepatitis C Virus (HCV) proteinexpression and replicationNAOUAL LINDA BENALI-FURET,1

RACHID FETOUCHI,1 RALF BARTENSCHLAGER,2

PATRICK MAUREL,3 ROZZARIO RIZZUTO,4

PATRIZIA PATERLINI-BRÉCHOT1

1INSERM/Pasteur U37O, Faculté de Médecine Necker-EnfantsMalades, Paris, France, 2Dpt of Molecular Virology, Heidelberg,Germany, 3INSERM U632, University of Montpellier 1,France, 4Dpt Experimental Medecine, University of Ferrara,Italy

The pharmacological control of HCV infection is a major issue. HCVproteins maturation and genome replication occurs into the ER, anorganelle known to be a key regulator of calcium signalling.We testedthe hypothesis that modulation of ER calcium concentrations in HCVreplicons expressing cells (Pietschmann et al, 2002) by specific drugsmay have an impact on HCV protein expression and genome repli-cation. By using a ER-targeted aequorin calcium probe we showedthat both cyclopiazonic acid (CPA) and 2-AminoethylDiphenylBorate(2-APB), induce a significant decrease of ER calcium concentrations(166 ± 10 μM, n = 5, p < 0.05 and 150 ± 10 μM, n = 5, p < 0.01respectively, vs 199 ± 20 μM, n = 7) in treated Huh-7 cells as com-pared to non treated cells. In HCV replicon expressing Huh-7 cells,western blot analyses showed that increasing concentrations of 2-APBand CPA induce a progressive decrease of NS3 (from 5% to 95%)and core (from 50% to 100%) proteins. Real time quantitative (RT)-

PCR specific to the HCV 5′ non-coding region revealed that con-centrations of 2-APB and CPA that do not modify cell viability inducea significant decrease of HCV RNA level of 65% and 90%, respec-tively. In agreement with these data, transfection of HCV repliconsexpressing cells with SERCA2 (which increases ER calcium concen-tration) led to an increase of HCV RNA level of 20%. These resultsshow that modulation of the ER calcium concentration can modulateHCV protein expression and HCV replication.

#216

High prevalence of HBV, HCV and HDV infection in MongoliaY DAHGWADORJ,1 G ZULKHUU,1

B BAYARMAGNAI,1 N CHOROLSUREN,1 B BAIGAL,1

G SARANGUO,1 O BAATARKHUU,1 D TUNGALAG1

1Health Sciences University of Mongolia

Background Viral hepatitis infection is hyperendemic in Mongolia.The objective of our study was to determine the prevalence of HBV,HCV and HDV infection in Mongolia.Patients and Methods The study is cross-sectional. We have ran-domly selected 1653 “healthy” subjects (0–80 ages) from randomized13 rural somons (county) of 12 aimags (province) and Ulaanbaatarcity. In all serum samples was determined HBsAg, anti-HCV andanti-HDV by ELISA.Results HBsAg detected in 187 (11.3%) and anti-HCV in 242(14.6%) of all serum. Anti-HDV in 60 [(38.5%) 4.3%] of 156 serum,which was detected HBsAg. But, there was markedly variability ofHBV and HCV marker depending on age groups. So, we estimatednumbers of carriers with chronic viral hepatitis infection by directstandardization method using population registered in the census2002. Then, now in Mongolia living approximately 281000 personwith HBV infection and 377500 person with anti-HCV or 70% ofthem 264000 person with HCV infection. They are more one a fifthof whole Mongolian population.Conclusion The epidemiological situation of chronic viral hepati-tis in Mongolia is catastrophic. Estimated numbers of HBV and HCVcarriers may use for hepatitis program in Mongolia.

#217

Evaluation of transfusion transmitted diseases inblood donors in Rasht, the capital city of Guilan, thenorth province of Iran from 2003 to 2005AZBARMI Z TAHERI,1 GHANAEI F MANSOUR,1

R JAFARSHAD,1 F JOVKAR,1 KH HAAJIKARIMIAN,1

S ALINEJAD,1 ESTAKHRI GH ABDOLLAHZADEH,1

SH NOURI1

1Gastrointestinal and Liver Diseases Research Center of GuilanUniversity of Medical Sciences

Background Transfusion transmitted disease (TTD) is a majorchallenge to the transfusion services all over the world. The problemof TTDs is directly proportionate to the prevalence of the infectionin the blood donor community. There are many blood borne, trans-fusion transmitted and related disease Such as hepatitis B/C, HIV,malaria, syphilis, cytomegalovirus virus, parvo-virus B-19 and bacte-rial infections.Materials and Methods A cross-sectional study was conducted toestimate TTDs among blood donors in Rasht, the capital city ofGuilan, the north province of Iran since 2003 till 2005. Hepatitis Bsurface antigen (HBs Ag) was tested by both enzyme-linkedimmunosorbent assay (ELISA) and Neutralizing HBsAb method, andanti hepatitis C virus antibody (anti HCVAb) was tested by both


enzyme immunoassay (EIA) and third-generation strip recombinantimmunoblot assay (RIBA), and human immunodeficiency virus(HIV) by both ELISA and Western Blot methods.Findings Of 49820 blood donors with mean age of 35.5 years, 225individuals (0.4%) had one of the TTDs, Including 130 subjects(57.7%) positive for HBs Ag, with both mentioned tests, 91 (40.4%)Anti HCV Ab positive persons, by both aforesaid methods, and 4cases (1.9%) positive for HIV Ab, confirmed by both mentioned tests.Of 225 subjects, 174 persons (77.3%) were married; 222 ones(98.6%) were males; 113 persons (50.2%) had less than a high schooleducation; 131 ones (58.2%) had free jobs; 85 individuals (37.8%)had positive O blood group; 156 persons (69.3%) donated blood atitinerant units of blood transfusion organization; 199 ones (88.4%)were Guilan native people; and it was the first time for 174 individ-uals (77.3%) to donate blood.Conclusions Persons who come to blood transfusion organizationare initially screened by some questions from staffs to identify highrisk individuals on the subject of their past medical history and lifestyles and inhibit them from blood donation if they are consideredhigh risk groups. In this study, TTDs had only 0.4% prevalenceamong blood donors, which is much lower than these diseases` preva-lence in Iran, that emphasizes the importance of such initial, easy, andinexpensive screening programs to prevent transmission of infectionsin patients receiving transfusions.

#218

Immunity against Hepatitis B virus in medicalvaccinated studentsF MANSOUR-GHANAEI,1 MS FALLAH,1 M ARAMI,1

F ABBASI,1 S NEMAT-DOOST,1 A ALE-ESMAEIL,1

M HOSENZADEH1

1Gastrointestinal and Liver Diseases Research Center (GLDRC)

Background All believe on hepatitis B as a burdensome viraldisease, that is the main cause of hepatic cirrhosis in developing coun-tries. 40% of Iran population has been exposed and infected by HBV.The risk of seroconversion after a needle-stick injury by HBs Ag-Positive blood is 7% to 30%. We know medical personnel especiallyurgency ward personnel who have frequent contact with blood andother HBV seropositive body fluids’ are at greatest risk. Anyway,Hepatitis B is the most important hepatic disease that can be pre-vented by vaccination. The evaluation of a group, which will play animportant role in future among health care staff, is a considerableduty. As the Anti-HBV medical students’ vaccination is so expensiveseroconversion evaluation by Anti-HBs tittering especially if there arefactors, which would arouse decreased immunologic response, seemsessential). So in this study we evaluated post vaccination immuno-logic response of Guilan university of Medical Science Medical students.Patients and Method All Students of Guilan University ofMedical Sciences, who had received complete HBV vaccine, wereincluded in this cross-sectional study. Demographic data and factorswere suspicious to influence the immunologic response, were col-lected. Anti-HBs Ab titer was measured with ELISA method. HBsAblevel equal or greater than 10mU/ml was considered as appropriateimmunologic response. Collected data was analyzed with SPSS 10.00software.Results From 443 medical students who were interviewed, 245met inclusion criteria and enrolled in the study with mean age of 24.9± 4.5 years (range: 20–53 years). 183 cases (74.7%) were female and62 cases (25.3%) were male. 233 cases (95.1%) had HBsAb equal orgreater than 10mU/ml and showed adequate response.While only 12cases (4.9%) didn’t respond properly to vaccination. Mean durationbetween the date of last vaccine and blood sampling in non-responsive group was higher than responsive group (P = 0.04).Immunologic response was significantly higher in females vs. males

(P = 0.001) and non-smokers vs. non-smokers (P = 0.02). Inadequateimmunologic responders’ mean age was 28.67 ± 5.4 years and 24.77± 4.4 years for adequate immunologic responders (P = 0.004).Conclusion Although HBV vaccination in medical studentsresulted in acceptable immune response in majority of cases, becauseof high exposure of this group to HBV infection, immunity againstHBV infection must be assessed 1–3 month after full three dose (0,1, 6) vaccination especially in men, elders and smokers.

#219

Comparison of real-time PCR assays for monitoringserum HBV DNA levels during antiviral therapyHAI-YING ZHANG,1 CHEE-KIN HUI,1 ANITA WONG,1

NIKKI P LEE,1 NANCY LEUNG,2 JOHN M LUK,1

GEORGE KK LAU1

1Queen Mary Hospital, Hong Kong, 2Department of Medicine,AHML Nethersole Hospital

Introduction & Aim Monitoring of serum HBV DNA levels isestablished as a gold standard to assess antiviral potency. However,currently used commercial assays have limitations with respect to sen-sitivity and dynamic range of quantification. The objective of thepresent study was to compare two real-time PCR based assays, theRealARTTM and Molecular Beacons assays, with the Digene HybridCapture II assay (ultrasensitive) in patients undergoing antiviraltherapy.Methods A total of 224 serum samples from 14 hepatitis B eantigen-positive chronic hepatitis B patients; 9 on adefovir dipivoxilplus emtricitabine combination therapy and 5 on adefovir dipivoxilmonotherapy, were tested for HBV DNA using the three assays.Results The RealARTTM and Digene Hybrid assays were signifi-cantly related (r = 0.94, p < 0.001). The Molecular Beacons andDigene assays were also correlated (r = 0.79, p < 0.001). There wasa good agreement between the RealARTTM and Digene assays. Themean difference was −1.05, as expected for good agreement, and 95%of the data were between +0.12 and −2.19 after logarithmic transfor-mation. The RealARTTM assay diverged from the Digene assay in arange of 2 orders of magnitude. On the other hand, the mean differ-ence was −1.73 when Molecular Beacons was compared with theDigene assay and the limits of agreement were +0.33 and −3.79respectively. Molecular Beacons differed from the Digene assay by 4orders of magnitude.The RealARTTM Digene and Molecular Beaconsassays revealed coefficients of variation of 5.33%, 6.03% and 6.47%,respectively.Conclusion The RealARTTM assay has a better correlation with theDigene assay and can be used effectively to monitor patients with highserum HBV DNA levels. It is also effective in the monitoring ofpatients whose serum HBV DNA levels have been suppressed toundetectable level by other commercial assays after antiviral therapy.

#220

Responses to peginterferon alfa-2a (40KD)(PEGASYS) treatment are durable in Asian patientswith HBeAg-negative CHB: 12 month follow-up datafrom a large randomised studyPATRICK MARCELLIN,1 GEORGE K.K. LAU,2

TEERHA PIRATVISUTH,3 RUI JIN,4 ZHI-MENG LU,5

MING-YANG LAI,6 SATAWAT THONGSAWAT,7

BALU RAMAKRISHNAN,8 MATEI POPESCU9

1Service d’Hépatologie, Hôpital Beaujon, University of Paris,Clichy, France, 2Department of Medicine, Queen Mary Hospital,University of Hong Kong, Hong Kong, China,3Songklanagarind Hospital, Prince of Songkla University,


Hat Yai,Thailand, 4Beijing You An Hospital, Beijing, China,5Department of Infectious Diseases, Ruijin Hospital, Shanghai,China, 6National Taiwan University Hospital,Taipei,Taiwan,7Department of Internal Medicine, Chiang Mai UniversityChiang Mai,Thailand, 8Roche, Dee Why, Australia, 9Roche,Basel, Switzerland

Background and Aims In patients with HBeAg-negative chronichepatitis B (CHB), treatment with peginterferon alfa-2a (PEGASYS)± lamivudine achieved significantly higher rates of response 6 monthspost-treatment versus lamivudine alone (Lau et al. NEJM 2005).Durability of response to PEGASYS in Asian patients participatingin a long-term follow-up study (LT-study) was evaluated.Patients and Methods In the original study, patients receivedPEGASYS 180 μg once-weekly for 48 weeks and and ALT normali-sation and HBV DNA suppression <20,000cp/ml were assessed 6months post-treatment. Patients were offered participation in the LT-study and were assessed 12 months post-treatment to see whetherresponse to PEGASYS treatment was durable.Results 85/108 Asian patients participated in the long-term study,of whom 46 (54%) had a virologic response (<20,000cp/ml) 6months post-treatment. Of these patients, 36 (78%) achieved HBVDNA levels ≤100,000cp/ml over the 6–12 month follow-up period:12 (26%) with HBV DNA <400cp/ml, 16 (35%) >400–<20,000cp/ml and 8 (17%) >20,000–≤100,000cp/ml. In total, 58 (68%) Asianparticipants in the long-term study achieved ALT normalisation 6months post-treatment. Of these ALT responders, 45 (78%) main-tained ALT levels >1–≤1.5 × ULN over the 6–12 month follow-upperiod: 36 (62%) with normal ALT and 9 (16%) with a peak ALTlevel >1 × ULN but δ1.5 × ULN.Conclusion More than 40% of participating Asian patients haddurable therapeutic response 6–12 months post-treatment. Nearly80% of patients who achieved sustained biochemical or virologicalresponses 6 months post-treatment maintained their response 12months post-treatment.

#221

Study on the association between the polymorphismof HLA class II gene and the outcome of infectionwith HBVYE-GUI JIANG,1 YU-MING WANG1

1Institute of Infectious Diseases. Southwest Hospital,ThirdMilitary Medical University, Chongqing 400038, China

Objective To investigate the association between the polymor-phism of HLA class II gene and the outcome of infection with HBV.Methods HLA-DRB1, -DQA1 and –DQB1 alleles in 52 patientswith chronic hepatitis B, 30 patients with acute hepatitis B and 106normal control subjects was analysed by using PCR/SSP. Cytotoxic-ity of TNF in Immortalized lymphoblastoid cell lines (LCL) wasdetermined by crystal violet staining. Lymphocyte proliferation index(PI) was detected by MTT.Results The allele frequencies of HLA-DRB1*0301, -DQA1*0501and -DQB1*0301 in the chronic hepatitis B group were markedlyhigher than that in the normal control group (X2

1 = 12.3068, Pc1 =0.0074. X2

2 = 9.2002, Pc2 = 0.0157. X23 = 15.5938, Pc3 = 0.0075).The

allele frequencies of HLA-DRB1*1101/1104 and -DQA1*0301 inthe chronic hepatitis B group were markedly lower than that in theacute hepatitis B group (X2

1 = 11.9206, Pc1 = 0.0145. X22 = 7.6781,

Pc2 = 0.0388). The cytotoxicity of TNF and PI in the HLA-DRB1*0301 and -DQA1*0501 and –DQB1*0301 positive groupswere markedly lower than that in the negative groups (t1 = 2.944, t2

= 3.397, t3 = 3.316; t4 = 5.865, t5 = 8.463, t6 = 2.974, P < 0.01). Thecytotoxicity of TNF and PI in the HLA-DRB1*1101/1104 and -DQA1*0301 positive groups were markedly higher than that in thenegative groups (t1 = 4.234, t2 = 4.873; t3 = 3.604, t4 = 3.860, P <0.01).

Conclusion HLA-DRB1*0301, -DQA1*0501 and -DQB1*0301are closely associated with the susceptibility to chronic hepatitis B,and HLA-DRB1*1101/1104 and -DQA1*0301 are closely associatedwith the resistance to chronic hepatitis B. HLA class II gene may takeeffect of immunological regulation through affecting TNF and affect-ing lymphocyte proliferation responce, and may be associated withthe chronicity of HBV infection and/or clearance of HBV.

#222

Hepatitis B virus quantitation and mutant detectionin clinical lab with Agilent 2100 BioanalyzerTONY KUNE,1,2 GUOCUI YANG,1,2 DAOPEI HUANG1,2

1Shanghai Haoyuan Biotech Co., 1998 Xin JinQiao Rd.Pudong, Shanghai China 201206, 2Agilent Technologies Inc.,395 Page Mill Rd., Palo Alto, CA 94306, United States

Abstract The PCR has been used for the detection of quantitationand mutant of HBV in clinical lab. Because of the PCR tube to tubevariation, an Internal Control molecule should be introduced in PCRfor quantitation.The YMDD mutant of HBV is a Lamivudin resistant strain, and the1896c mutant of HBV is a strain which inhibits HBeAg expression.Analysis of these four PCR products, including HBV, IC, YMDD,1896c, is labor intensive. “Lab-on-a-chip” (including Agilent 2100Bioanalyzer and associated chip & reagents) offers an integrated fluidhandling, sample processing, separation and detection in a miniaturechip format. This technical platform is applied to analyze 4 PCRproducts of HBV (Fig 1). It brings greater efficiency and cost savingsto clinical labs.

#223

HBV genes induce cytotoxic T lymphocyte responseupon adeno-associated virus (AAV) vector deliveryinto dendritic cellsHONG YOU,1 PING WANG,1 MIN CONG,1

TIAN HU LIU1

1Beijing Friendship Hospital

Objective Hepatitis B virus (HBV) has been an expanding problemthroughout the world and remains difficult to treat. Immunothera-peutic approaches may offer new, effective treatments. The study isto show that multiple HBV genes delivered by recombinant adeno-associated virus (AAV) virus into DC are able to elicit a strongantigen-specific and MHC class I-restricted CTL response.Methods Three recombinant AAV type 2vectors, carrying eitherthe HBV S, C or X gene, were used to transducer professional antigenpresenting dendritic cells (DC) for the purpose of stimulating


cytotoxic T lymphocytes (CTL) in vitro. Monocytes were isolatedfrom 10 healthy donor and 10 chronic hepatitis B patients with highHBV DNA level. Monocytes were pulsed by rAAV-HBV-S, X, C or293 lysate as control at the first day of isolation, then the dentriticcells were cultures for 7 days in vitro. The transcription and expres-sion of HBV- S, C or X gene were analyzed by reverse transaction—polymerase chain reaction (RT-PCR) or intracellular stainingfluorescence actived cell sorter (FACS) respectively.Results It was found that all tree recombinant AAV/HBV antigenvirus loaded DC at approximately 90% transduction efficiency. Mostimportantly, all three AAV-loaded DC stimulated rapid, antigen-specific major histocompatability complex (MGC)-restricted CTL. Invitro these CTL killed (30–50%) synthetic antigen-positive autolo-gous targets as well as Hep3B liver cell targets. In comparing the threeantigens it was found that AAV/HBV-C-derived CTL consistently hadthe highest killing efficiency. Further studies showed that AAV/HBV-C-derived CTL had higher IFN-gamma, and low IL-4 expression.Conclusion These data suggest that AAV/HBV antigen gene-loading of DC may be useful for immunotherapeutic protocols againsthepatitis B virus infection and that the HBV C antigen may be themost useful for this purpose.

#224

Studies on the relationship between hepatocellularcarcinoma and HBV genotypes in Guangdongprovince of ChinaYUEHUA HUANG,1,2 ZHANHUI WANG,1 SHIWU MA,1

JINLIN HOU1

1Hepatology Unit and Department of Infectious DiseasesNanfang Hospital, Southern Medical University, 2Department ofInfectious Diseases, Guangzhou Medical Collegee

Objective To study the relationship between hepatocellular carci-noma and HBV genotypes in GuangDong province.Methods We used a polymerase chain reaction-restriction fragmentlength polymorphism (PCR-RFLP) method to determine HBV geno-types in 298 patients with hepatocellular carcinoma and chronichepatitis B.Results The genotype distribution for 298 patients with chronicHBV infection and hepatocellular carcinoma was as follows: B, 172(57.7%); C,126 (42.3%); other genotypes have not been found. Thedistribution of genotype C is the highest in patients with hepatocel-lula carcinoma than other groups (P < 0.01). Compared with maleand female patients, the incidence of male gender was significantlyhigher than that of female gender in all patients. Also, comparing withthe distribution of age shows that older age are more significant inhepatocellular carcinoma and liver cirrhosis groups than the otherones. Thus, the evident risk factors for advanced liver disease andhepatocellular carcinoma were old age, male gender and genotype C.Conclusion B and C genotypes are the main types in HBV relatedhepatic disease in Guangdong district, while C genotype is morecommon than B genotype in patients with HCC.

#225

Suppression of tissue inhibitor of metalloproteinase-1(TIMP-1) gene expression by recombinant adeno-associated virus carrying small interference(siRNA) of TIMP-1 in rat hepatic stellate cell (HSC)MIN CONG,1 WANG PING,1 TIAN-HUI LIU,1

SHU-ZHEN TANG,1 BAO-EN WANG,1 JI-DONG JIA,1

YONG LIU,1 HONG YOU1

1Liver Research Center, Beijing Friendship Hospital, CapitalUniversity of Medical Sciences

Objectives Elevated tissue inhibitor of metalloproteinase-1(TIMP-1) expression contributes to excess extracellular matrix in liver fibrosis. The study was designed to construct recombinant adeno-associated virus (AAV) carrying siRNA of TIMP-1 and investigate thelong-term effect of TIMP-1 gene RNA interference on rat hepaticstellate cell (HSC)-T6 cells in vitro.Methods Five siRNA oligomers targeting rat TIMP-1 at nt161–183, nt 190–208, nt 208–226, nt 226–244 and nt 445–463 weretransfectedinto rat HSC-T6 cells respectively. U6 promotor followedby the annealing siRNA which had the strongest suppression effectwere cloned into the AAV vector and packed in 293 cells to constructthe recombinant AAV/siRNA-TIMP-1/neo. After infectiong thisrecombinant AAV into rat HSC-T6 cells and selection by G418, real-time PCR after reverse transcription and Western blot were performedto detect the transcription and expression level of TIMP-1 gene inHSC-T6 cells at 1 and 3 month.Results It was demonstrated that 2 of the 5 siRNA oligomers hadthe significant effect on suppressing TIMP-1 expression by Western-blot within 72 hours after transfecting into rat HSC-T6 cells. Theamplified rat U6 promotor by PCR using the DNA of infected cellsby the recombinant AAV/siRNA-TIMP-1/neo as template proved thatthe HSC-T6 cells could continually express siRNA-TIMP-1 for 3months after infection. The transcription and expression level ofTIMP-1 in HSC-T6 cells which were infected by the recombinantAAV were suppressed dramatically compared with mock control andnormal HSC-T6 cells (P < 0.05), and the expression level of TIMP-1 gene in HSC-T6 cells infected after 3 months decreased signifi-cantly (90%) compared with those infected after 1 months (60%).Conclusion RNA interference can exert an suppression of TIMP-1 gene in rat HSC, and when this fuction combined with AAV infec-tion, it can suppress the specific gene expression for a long time bychromosomal integration.

#226

Prognostic factor analysis and modeling for patientwith chronic severe hepatitisYU-MING WA,1 NAN ZHANG,1 GUO-HONG DENG1

1Department of infectious diseases, Chongqong southwest Hospital

Objective to analyze the prognostic factor and develope a prog-nostic model for patients with chronic severe hepatitis (CSH).Methods 385 patients admitted to hospital from December, 1998to October, 2003 for the treatment of chronic severe hepatitis wereevaluated. The main clinical and laboratory variables were analyzedas predictive factors of survival by Cox univariate and multivariateregression model.Results The median survival time of this study group is 47 days.The survival rates was 66.23%, 32.86%, 26.9% at 1, 3, 6 monthsrespectively, 22.97% at 1-yea, and 17.67% at 3 years. We observeddifferent prognostic factors between patients with or without liver cir-rhosis. For patients with decompensated liver failure, prognostic index(PI) = 0.561 *HE-0.053*PTA; for patients with non-deconpensated

Tab. Recommendation of the nomenclature of liver failure

Nomenclature Definition

ALF* Acute onset, with liver failure in 2 weeksSALF* Subacute onset, with liver failure in 15–168 daysACLF* Acute liver decompensation on the basis of end stage

liver diseaseCLF� Chronic liver decompensation on the basis of end

stage liver disease

*With or without chronic liver disease, can be devided into 2 subtypes:HE and non-HE� T.Bil < 171 μmol/L, the other types: T.Bil � 171 μmol/L


liver failure, the PI = 0.001*AST-0.042*Albumin + 0.01* TBIL-0.029*PTA + 1.117*HE. Our model accurately predicted 3 monthssurvival on an independent series of 84 patients with chronic severehepatitis.Conclusion The model is valuable on prognostic evaluation ofCSH patients and may be useful to guide clinicians in selection oftreatment method for CSH.Key word chronic severe hepatitis; prognosis; Cox model

#227

HBsAg and HBsAb coexistence in Chinese patientswith chronic HBV infection: clinical and virologicalcharacteristicsYUE-CHENG YU,1 SHI-WU MA,1 ZHAN-HUI WANG,1

XUE-GANG WANG,1 MIN-FENG LIANG,1

YAN-JUN WANG,1 YA-NAN SONG,1

GUANG-WEN ZHANG,1 KANG-XIAN LUO,1

JIN-LIN HOU1

Hepatology Unit and Infectious Diseases Department ofNangfang Hospital, Southern Medical Univeristy, GuangzhouCity 510515, China

Backgrounds Generally, the surface of Hepatitis B Virus (HbsAg)and corresponding antibody (HbsAb) can not be detected at the sametime. But with the development of sensitive detection assays, AbbottAXSYM system, a microparticle enzyme immuno-assay (MEIA),increasingly number of Chinese patients with chronic HBV infectionwith coexisted HBsAg/HBsAb were recorded. However, significanceremains to be determined.

Methods and results 1.87% (88/4,701) cases with chronic HBVinfection were found to be HBsAg/HBsAb coexistence, and the resultswere confirmed by repeated test. 4.78% (291/6,087) cases wereHBeAg/HBeAb double-positive, and this is significantly higher thanthat of HBsAg/HBsAb coexistent ones (P < 0.01). HBsAg ≥200S/Nor HBsAb ≥ 100 mIU/ml was regarded as the high titer, thus theHBsAg/HBsAb coexistence could be divided into 4 types: 39.78%cases were high HBsAg and low HBsAb, 13.98% were high HBsAgand high HBsAb, 37.63% were low HBsAg and low HBsAb, 8.6%were low HBsAg and high HBsAb. The age covered from 11 to 77years old (median 36).The ratio of female cases was 26.14% (23/88),significantly higher than 17.17% (340/1640) of controls (P < 0.05).Fifty-three cases having recorded clinical informations were furtheranalyzed. Among them, 16.98% had received standarded antiviraltherapy with interferon alpha or/and lamivudine in recent two yearsbefore detection. Fifty out of 88 cases with HBsAg/HBsAb coexis-tence had HBV DNA quantification results. 56.14% were less than 1× 105 copies/mL, and 43.86% were more than 1 × 105 copies/mL. InHBsAg/HBsAb coexistent cases, the constituent ratios of HBV geno-type B, C, or B + C were 64.91%, 29.82% and 5.26%; but in controlgroup (n = 267), the ratios were 52.81%, 46.07%, and 1.12%, respec-tively (P < 0.05). Several point mutations were found in surface geneof HBV, especially in the hydrophilic regions within/outside ‘a’ deter-minant of HBsAg.Conclusions HBsAg and HBsAb can exactly exist in the samesamples, and might be more apt to occur in patients with HBV geno-type B, low HBV DNA replication and female. Mutation of HBsAgis one of the important explaination, but may not be the only mech-anisms. The host’s immue pressure might become the causes moreoften than antiviral pressure.

Immunization of a recombinant plasmid encoding hepatitis C virus core protein

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