Tuesday Posters: HBV Pathogenesis May 23rd 8:00 AM-5:00 PM
A. Yasuo ; M. Moriyama; A. Yasuyuki
The purpose of this study is to assess the influence of occult HBV infection on histopathologic impact and its clinical outcome of chronic hepatitis C according to prospectively, and is to determine the nucleotide sequence of occult HBV infection.
Materials and Methods.
The subjects were 468 paients with HCV RNA-positive (C-viral) chronic hepatitis (CH) or liver cirrhosis (LC) who were negative for serum hepatitis B surface (HBs) antigen, and underwent liver biopsy from, l993 to 2003 The serum hepatitis B virus (HBV) DNA was measured by the nested PCR using the primer set of the X gene region. HBV DNA-positive patients and -negative patients were compared with clinical profiles, histological findings and long-term outcome. Then, the serum HBV DNA level of 182 out of 204 patients sera whose serum HBV DNA was detected by nested PCR were measured using the real-time detection direct test. Further, we determined the nucleotide sequence of 2nd PCR products, and analyzed HBV genotype by phylogenetic tree anlysis.
The serum HBV DNA was detected in 204 (43.6%) of the 468 patients studied. There were no significant differences between HBV DNA-positive patients and -negative patients concerning clinical profiles. HBV DNA-positive patients of the degree of the inflammatory cell infiltration and irregular regeneration of hepatocytes showed significantly stronger than those of HBV DNA-negative patients. Serum HBV DNA levels in 169 patients sera (91.8%) showed less than 1.0 log IU/ml. 15 patients sera whose HBV DNA levels more than 1.0 log IU/ml, and were classified 11 for 1.0 to 1.9 log IU/ml, and others were 2.1 log IU/ml, 3.0 log IU/ml, 3.5 log IU/ml and 5.3 log IU/ml. Cumulative probability of hepatocellular carcinoma (HCC) incidence of HBV DNA-positive patients were significantly higher than HBV DNA–negative patients. Furthermore, HBV DNA-positive was risk factor for occurrence of HCC from C-viral liver disease by multivariate regression analysis. Alignment of the nucleotide sequences of HBV genotypes A to F and the 6 NU isolates revealed the nucleotide substitutions nt 12 in C to T, and nt 14 in G to A. The HBV isolates from these patients were classified as HBV genotype C by phylogenetic analysis. The HBV isolates from these patients were clustered in the same group as isolates from the Japanese mainland.
The histopathologic impact and long-term outcome of C-viral CH or LC could be affect by occult HBV infection. The HBV isolates from these patients were classified as HBV genotype C
T1855. Mutations of hepatitis B virus surface gene on T cell epitopes in children with chronic infection and hepatocellular carcinoma.
Y. H. Ni; M. Chang; H. Hsu; H. Chen
Hepatitis B surface antigen (HBsAg) is an antigen the host immune response attack against in hepatitis B virus (HBV) infection. This study aims to investigate the mutation(s) of HLA-A2 restricted T cell epitope (TCE) on HBsAg and its relation to chronic HBV infection. Totally 441 carrier children were followed-up for more than 10 years. TCE mutation was examined at the latest follow-up. They were divided into hepatitis e antigen (HBeAg) (-) (n=60) and HBeAg (+) groups based on their initial status. HBeAg (+) group was further divided into HBeAg (+/+) group (n=152) and HBeAg (+/-) group (n=229) if spontaneous HBeAg seroconversion occurred. Another 25 children with HBV-related hepatocellular carcinoma (HCC) were also enrolled. The TCE mutations, liver enzymes, genotypes, HLA, and vaccination histories have been correlated. HBeAg (+/-), HBeAg (-), and HCC groups had a similar TCE mutation rate, (26.7%-29.2%), which was higher than that of HBeAg (+/+) group (7.9%). In HBeAg (+/-) group, 11.9% (8/67) already had such mutations existing before HBeAg seroconversion. Ten mutations spanning in codons 31-51 of HBsAg were found, while codons 40 and 44 were two hot spots. Those with TCE had an older age of HBeAg seroconversion than those without (16.1±5.3 vs. 12.7±5.7 years p<0.0001.) Sixty-six of the 441 subjects received HBV immunization but failed. TCE mutation rate was higher in the unimmunized patients than the immunized patients. Genotypes and HLA did not play major roles in the emergence of TCE mutation. In conclusion, the TCE mutations in children may develop before HBeAg seroconversion, but it mostly occurred after HBeAg seroconversion.
J. Wang; H. Wang; C. Zhu
To investigate the relationship between clinical/pathological features and development of cirrhosis in patients with chronic hepatitis B (CHB). To determine the time to progress(TTP) and the prognostic risk factors for developing cirrhosis in patients with HBeAg negative CHB (e-CHB) compared with HBeAg positive CHB (e+CHB).
Data collected in consecutive CHB patients confirmed by liver biopsy (informed consent signed)within 1 week after hospitalized in Zhongshan Hospital, Shanghai from May, 1993 to Jan. 2004 and followed up until June, 2004. Patients received antiviral treatment were excluded. Scheuer classification has been used for grading and staging of liver specimen by one pathologist. The difference of grade and stage, and difference of TTP between e-CHB patients and e+CHB patients were assessed by rank sum test and life table with Gehan test respectively. Cox regression model was used to determine the prognostic risk factors associated with developing compensated and decompensated cirrhosis.
434 patients met the inclusion criteria, 52 (12%) lost follow up. 382 patients (male 315 and female 67; age 15-71 yr with mean 33.57 yr) were included in this study. After the follow-up period of an average of 88.64 weeks (20-260 weeks), 75 cases (19.6%) occured compensated cirrhosis with an annual occurrence rate of 0.95%. 32 cases (8.4%) progressed to decompensated cirrhosis with an annual occurrence rate of 0.39%.
120 patients (M 106, F 14 ; age: 34.3yr) were e-CHB, 262 were e+CHB (M 209, F 53, age 32.93yr). The cases developed compensated cirrhosis in e-CHB group compared to e+CHB was 22 (22.5%) vs 48 (18.3%) respectively. The cases progressed to decompensated cirrhosis were 14 (11.61%) in e-CHB group and 18(6.87%) in e+CHB group respectively. There is no statistically significant difference between these two groups for grade and stage by rank sum test as well as cirrhosis development showed in life table by Gehan test. The result of multivariable analysis by Cox regression model also indicates that histological staging at hospitalization is independent prognostic factor for developing compensated and decompensated cirrhosis in CHB (P<0.01, HR: 2.14 and 1.7). Histological grade, serum albumin, globulin, AST, ALT, AFP, PT tested at hospitalization, age, sex, history of HBV, e-CHB were not associated with development of cirrhosis in this model.
The severity of the fibrosis stage at presentation is closely related with compensated and decompensated cirrhosis development in CHB rather than severity of inflammatory grade and e-CHB.
T1857. HBV vaccination with evidence of protective antibodies titer induces CD4+/CD25+ T cells
A. Perrella; L. Racioppi; L. Atripaldi; S. Pisanti; C. Sbreglia; P. Bellopede; O. Perrella
The immunoregulatory mechanisms occurring after the HBV vaccine administration are not well known. Aim of the present study was to enumerate CD3+/CD4+/CD25+ T cells and to test their function after HBV vaccination at the seroconversion of HBsAg comparing them to patients with chronic hepatitis B.
We enrolled 10 subjects undergoing three doses of HBsAg vaccine (Engerix-B DNA-recombinant)injected intramuscularly in the deltoid region at time 0(T0),one(T1) and sixth month(T6), following the manufacturer’s instructions (Group A) and 10 healthy donors (Group B) (table). CD3+/CD4+/CD25+ lymphocytes were enumerated in PBMC using flow cytometry (Beckman Coulter). T lymphocytes activity was assessed using immunomagnetical sorting with magnetic bids (purified Tregs > 95%) (Dynal Treg kit) as capacity to inhibit the proliferation of CD4+/CD25- T cells stimulated with anti-CD3/CD28 (1:2 and 1:20 ratio).
Subjects were administered vaccine after one month achieved a protective titer of Anti-HBs (> 100IU/mL) and had a contemporary statistically significant increase of CD4+/CD25+ frequency and function when compared to patients suffering chronic hepatitis B and their self before the administration of first dose of vaccine (U Mann-Whitney Test z= 2.23; p < .05 and z= 2.22; p< .05)(table).
An increased CD3+/CD4+/CD25+ T cells frequency and function seems to occur after one month of vaccine administration when a protective titer is achieved, suggesting a role of CD4+/CD25+ T regulatory cells in the seroconversion and proposing them as a possible tool to monitor antiviral treatment response.
T1858. Novel Subgenotypes of Hepatitis B Virus Among Chronic Liver Disease Patients in the Philippines
T. Sakamoto; Y. Tanaka; E. Orito; J. Co; J. Clavio; F. Sugauchi; K. Ito; A. Ozasa; A. Quino; R. Ueda; M. Mizokami; J. Sollano
Several hepatitis B virus (HBV) subtypes (subgenotypes), HBV/Aa (Asia/Africa), Ae (Europe), Ba (Asia) and Bj (Japan) were reported with respect to the clinical differences between the patients infected with these subtypes. There have been no investigative population studies on HBV genotypes in the Philippines. We investigated the HBV genotype distribution among patients with chronic liver diseases (CLD) in the Philippines. One hundred sera were obtained from CLD patients, consisting of 32 chronic hepatitis (CH), 37 cirrhosis and 31 hepatocellular carcinoma (HCC). Nine complete genomes and 100 core promoter/precore genes of HBV were sequenced directly Phylogenetic analyses showed that 51 HBV/Aa, 22 HBV/Ba and 27 HBV/C strains were found in this study. Interestingly, most HBV/C strains in the Philippines had the specific cluster distinct from previous HBV/C strains (C1-4), indicating a novel subtype, HBV/Cp (p standing for Philippines; C5). Moreover, most HBV/B strains had the specific cluster as the fifth HBV/B subgroup (B5) with viral characteristics of HBV/Ba. In the 3 genotypes, HBV/Ba or HBV/C were significantly more prevalent in cirrhosis and HCC patients than HBV/Aa (p<0.02). The prevalence of core promoter (CP) mutation (T1762/A1764) was more frequent in HCC patients with HBV/Ba and HBV/C. Multivariate analysis indicated that age (OD 3.43; 95%CI 1.04–11.36; p=0.044) and CP mutation (OD 14.08; 95%CI 3.62–4.74; p<0.001) were significant factors for HCC development. In conclusion, novel subtypes Cp and B5 are prevalent in the Philippines, as well as HBV/Aa.
T1859. Association between Hepatitis B Virus Infection and HLA-DRB1 Genotyping in Shaan’xi Han patients in Northwestern China
J. Liu; G. Yang; H. Xie; S. Han; R. Du; D. Fan
HBV infection is a major public health problem worldwide, especially in China, where 7±20% of the large population is chronically infected with estimated millions. The mechanism of susceptibility to chronic persistent hepatitis B virus (HBV) infection is not well clarified, while the outcome of HBV infection mainly depends on the host immune response. HLA class II molecule is an integral component of the immune response on which majority of host genetic studies have concentrated. Many different HLA class II alleles have been demonstrated to play roles in the HBV infection. In this study, the association of HBV infection and HLA-DRB1 allele in Han individuals in Western China population were studied for the first time.
216 Shaan’xi Han individuals were categorized into 2 different groups: the HBV patients group (n=108), and health control group (n=108). HLA Class II types were determined by PEL-FREEZ® SSP UniTray® which is a PCR-based method designed to provide low to high resolution of the various HLA types.
DRB1*04, DRB1*09, DRB1*12, DRB1*15 were the most common genotypes in all groups with the frequencies of 16.2%,12.5%,11.6% and 13.4% respectively. The allele frequencies of HLA-DRB1*03 (10.6% of HBV patients versus 3.7% of health controls, odds ratio=3.10; Pc=0.008; P<0.05) and HLA-DRB1*07 (17.6% of HBV patients versus 9.3% of health controls, Pc=0.016; Odd ratio=2.09; P<0.05) were markedly higher in HBV group. The allele frequency of HLA-DRB1*15(6.9% of HBV patients versus 13.4%of health controls Pc=0.039; Odds ratio=0.48; P<0.05) was obviously lower than that in controls. It is indicated that HLA-DRB1*03 and HLA-DRB1*07 are related with susceptibility to chronic hepatitis B, and DRB1*15 is closely related with resistance to chronic hepatitis B in Northwestern China. Similar results was got on DRB1*03 and 15 alleles between the patients (n=108) and 46 HBV seronegative spouses of HBV patients, which was a high-risk group for HBV infection.
All these suggest that host HLA classII gene is an important factor determining the outcome of HBV infection.
T1860. Hepatitis B genotype Bj in a cohort of Alaska Native People with chronic infection.
J. Simonetti; M. Snowball; S. Negus; C. Homan; J. Williams; Y. Tanaka; M. Mizokami; B. McMahon
Hepatitis B (HBV) has been classified into eight genotypes (A-H) with distinct geographical distributions and differing influences on the outcome of chronic infection. Recently, recombination between genotypes has been reported and is emerging as an important factor in the outcome of HBV infection. Further investigation has revealed that the Taiwanese genotype B (or Ba) harbors a recombination with genotype C over the precore region plus the core gene while the Japanese B (or Bj) does not harbor a recombination and has a less severe disease course. Bj is found exclusively in Japan while Ba is ubiquitous in the other countries in Asia. In addition, HBeAg positivity and the basal core promoter mutation (T1762/A1764) are found to be more frequent in Ba than Bj. A long-term study of Alaska Native People with chronic HBV infection has identified 5 genotypes with genotype B constituting 4% (46/1188) of the total number. In this preliminary study we further identified the subtype of genotype B in this cohort.
Genotypes Ba and Bj were identified through semi-nested PCR and direct sequencing of a 700-bp region of the precore region plus the core gene. Serum with sufficient HBV DNA was available for 40 of the 46 original genotype B patients. HBV DNA levels were determined using a Real-time Quantitative PCR assay.
Of the 40 genotype B patients tested 100% were classified as Bj. The median age of the genotype B patients is 55 and to date hepatocellular carcinoma (HCC) has been diagnosed in only one individual (a 76-year old male) from this group and none have been diagnosed with chronic hepatitis. Furthermore, the genotype B patients in this cohort maintain consistently low (< 20,000 IU/ml) HBV DNA levels and rarely have elevated liver function tests.
Genotype Bj is the exclusive genotype B subtype found in a cohort of Alaska Native People with chronic HBV. In addition, the disease course of patients infected with genotype Bj mirrors the less severe course observed in Japan.
J. M. Mellen; N. L. Kyulo; V. Xia; J. Hoefs; D. Imagawa; K. Hu
The clinical course of HBV infection varies with ethnicity. Although numerous studies have focused on the natural history of this disease in the Asian population, little is known about the clinical presentation of chronic HBV infection in Asian Americans.
To define the natural history of HBV infection in Asian Americans.
This was a retrospective study by chart review of > 300 consecutive patients presented to the University of California Irvine Medical Center between Jan. 1990 and Nov. 2005 with a diagnosis of HBV infection. The inclusion criteria were documented Asian ethnicity, positive HBsAg or HBV DNA for 6 months, no prior HBV treatment at presentation, and no HCV or HIV co-infection. After excluding 140 cases who did not meet the inclusion criteria, 172 patients were included in the study.
This cohort of patients had a mean age of 41.9 + 15.0, contained 66.9% male patients, 97.8% were born outside of the U.S., and 13.4% had histological diagnosis of hepatocellular carcinoma (HCC). Family history of hepatitis B was reported in 47.7% of the patients, but history of injection drug use (2.5%), blood transfusion (14.4%), and current alcohol use (12.1%) were less common. Of the 57 patients with liver biopsy, 42.1% had stage 3-4 fibrosis. In 14 patients with HCC, stage 3-4 fibrosis was present in 78.6% of non-tumorous liver tissue, and this was significantly higher than in patients without HCC (30.9%, p=0.004). Stage 3-4 fibrosis was not significantly associated with the baseline ALT value (p=0.76), HBV DNA load (p=0.78), steatosis (p=0.21). The mean body mass index (BMI) was 23.8 + 3.4 kg/m2, and steatosis was reported in 13.0% of the patients. The incidence of HBeAg negativity was as high as 79.5% in this cohort of patients. HBeAg-negative HBV infection was associated with elder age (p=0.05), no known family history of hepatitis B (p=0.009), non-elevated ALT (p=0.001), and a lower HBV DNA load (p=0.0003). However, HBeAg status was not associated with steatosis (p=0.56), stage 3-4 fibrosis (p=0.45), or a diagnosis of HCC (p=0.46). A multivariate analysis indicated that after adjusting for age and family history of hepatitis B, a low HBV DNA load and a non-elevated ALT were independently associated with HBeAg-negative HBV infection.
This single center study indicated that HBeAg-negative HBV infection is a common presentation in Asian Americans, and that it is independently associated with a low HBV DNA load and a non-elevated ALT. Asian American patients with chronic HBV infection carry a high incidence of advanced fibrosis and a low incidence of steatosis.
T1862. Occult hepatitis B virus infection with and without anti-HBs antibodies - implications in the development of hepatocellular carcinoma C.
Tan; J. Chang; G. Lim; Z. Yi; L. Ooi; P. Chow; A. Chung; W. Chow
Occult hepatitis B virus (OHBV) infection is defined as the detection of HBV DNA in serum or liver tissue of HBsAg negative patients. Patients with OHBV infection who are anti-HBc positive can be further differentiated into those with and without anti-HBs antibodies (anti-HBsAb). Those with anti-HBsAb may be regarded as having evidence for immunoclearance of HBsAg and hence have an advantage over those without anti-HBsAb who are often regarded as low grade viraemic carriers. Hepatocellular carcinoma (HCC) uncommonly occurs in cases of OHBV infection and HBV DNA may be detected in the liver and tumor tissues. However, it is not known whether the extent of tissue HBV integration differs between cases with and without anti-HBsAb.
To study whether the extent of HBV integration in tumor and non-tumor tissues is influenced by presence of anti-HBsAb in HCC patients with occult HBV infection.
Study subjects were consecutive HBsAg-negative, anti-HBc positive HCC patients who underwent surgical resection at our centre. Our IRB approved the study and all patients gave informed consent. Patients were grouped based on anti-HBsAb status – Group 1 (anti-HBsAb positive) and Group 2 (anti-HBsAb negative). Nested PCR techniques were used to determine presence of HBV “S” and “X” genomes in tumor and non-tumor tissues and serum. Ten HBsAg positive and 5 seronegative (HBsAg/anti-HBc/anti-HBs/serum HBV DNA) patients with resected HCC served as positive and negative controls respectively.
There were 6 patients in Group 1 and 5 in Group 2. Mean anti-HBsAb level in Group 1 was 134iu/ml (range 17-564). “S” genome was detected in 5/6(83%) tumor tissue (TT) and 6/6(100%) non-tumor tissue (NT) in Group 1 and 4/5(80%) TT and 3/5(60%) NT in Group 2. “X” genome was detected in 3/6(50%) TT and 6/6(100%) NT in Group 1 and 5/5(100%) TT and 3/5(60%) NT in Group 2. Concordance rates between NT and TT in detection of “S” genome were 83% and 80% for Groups 1 and 2 respectively; for “X” genome, they were 50% and 60% respectively. There were no significant differences (Fisher’s test) between the 2 groups in all the above results. As for the sera, nested PCR did not detect “X’ genome at all in both groups but “S” genome was found in 1 case in Group 1 that also had integration of “S” and “X” genomes in both TT and NT.
Despite the presence of anti-HBs antibody, there is a similar extent of genomic integration of HBV into host liver tissue. This suggests the pathogenesis of HCC is similar in OHBV infection with or without anti-HBsAb. Therefore, regardless of anti-HBsAb status, strategies to prevent the development of HCC should be similar.