Hepatitis B virus and hepatocellular carcinoma

Hepatitis B is a very important public health problem. Epidemiologic studies have shown a relationship between the hepatitis B virus (HBV) chronic carrier state and the development of hepatocellular carcinoma. HBV belongs to the Hepadna viruses family which includes the woodchuck hepatitis virus (WHV), Woodchucks infected with WHV represent a good experimental model to study the viral oncogenesis. In 85% of the studied cases, WHV acts by insertional mutagenesis in a gene of the myc family, mostly the N-myc2 gene. Expression of the myc genes is increased, suggesting the role of the viral enhancer. Study of transgenic mice have shown the liver specificity of the WHV action. In man, the liver oncogenesis is not explained. Studies are in progress to detect inactivation of tumor suppressor genes.     

Isolation of an ovine genomic sequence containing the full-length angiotensin II type-1 receptor

We have isolated from a genomic library using PCR amplification an 1171 base sequence containing a putative ovine AT1-R protein coding sequence of 1080 bases. As expected the protein coding sequence is of greater than 99% homology to the partial protein coding sequence reported by Robillard et al, with only one base difference. Relative to other species, highest homology at the level of the cDNA protein coding sequence is to bovine (97.6%) and lowest homology to rat Type 1a (83.3%). The predicted protein amino acid sequence in turn encodes a protein with the properties of a seven alpha-helix transmembrane receptor (by TMPred) sharing closest homology (98.6%) to the bovine receptor and lowest to the rat Type 1a (90.2%). As expected from such a high degree of interspecies homology, amino acids identified by site-directed mutagenesis of the human or rat AT1A-R as involved in binding and action of AII are very highly conserved in the ovine sequence. In addition, both bovine and ovine AT1-R are known to exhibit lower affinity for DuP753 than human AT1-R, and in bovine AT1-R this has been suggested to coincide with the amino acid substitutions Ala->Thr (163) and Leu->Met (262) relative to the human sequence. Our ovine AT1-R cDNA sequence shares these same bovine substitutions.     

Hepatitis B virus genomic heterogeneity: variation between quasispecies may confound molecular epidemiological analyses of transmission incidents

Nucleotide sequence variability studies were conducted on a 263-base pair fragment of the core-coding genomic region of hepatitis B virus (HBV), amplified by the polymerase chain reaction (PCR) from three surgeons with varying circulating levels of HBV, all of whom were thought to have transmitted HBV to their patients post-surgically. DNA sequencing was applied to amplicons obtained directly from serum and those cloned into plasmid vectors, and from single HBV molecules in serum separated by a limiting dilution procedure. In one surgeon, who had a titre of approximately 3 x 10(5) genome equivalents ml-1, the direct sequence was identical to none of 29 other sequences and differed by one base substitution from the sequence amplified from the single patient he infected. In another surgeon, who had a titre of approximately 2 x 10(6) genome equivalents ml-1, the direct sequence was identical to 17 of 36 (47%) sequences; however, the sequence common to all three infected patients was identical to a unique sequence in the surgeon that differed by three base substitutions from the direct sequence. By contrast, the direct sequence in the third surgeon, who had a titre of approximately 4 x 10(7) genome equivalents ml-1, was identical to 25 of 38 (66%) sequences, and to the sequence common to all 11 infected patients. Assessment of HBV DNA sequences directly amplified from clinical specimens may not be appropriate to studies of transmission in which the source of infection harbours a relatively dilute, heterogeneous mix of viral variants.     

Differences of resected hepatocellular carcinoma with hepatitis B or C virus

BACKGROUND/AIMS: The purpose of this study was to clarify the clinicopathologic differences of hepatocellular carcinoma associated with the hepatitis B versus the hepatitis C virus. METHODOLOGY: One hundred and sixty-eight patients with resected hepatocellular carcinoma were tested for viral hepatitis. Ten (6%) had both the hepatitis B surface antigen and antibodies to the hepatitis C virus. Thirty-three (20%) had neither marker. Sixteen (9%) had only the hepatitis B surface antigen (group B), and 109 (65%) had only antibodies to the hepatitis C virus (group C). We compared groups B and C clinicopathologically. RESULTS: The mean tumor diameter was larger in group B than in group C (6.3 cm vs 3.4 cm), while group B patients were younger than group C (48 yrs vs 62 yrs, p<0.0001). Poor liver function, histologic cirrhosis and chronic active hepatitis were frequently found in group C. The 1- and 2-year tumor-free survival rates following surgery in group B were 67% and 33%, and those in group C were 73% and 49%. The 1-, 2-, and 3-year survival rates following surgery in group B were 78%, 68%, and 0%, while those in group C were 92%, 83%, and 76% (p=0.0189). CONCLUSIONS: Hepatocellular carcinoma with concomitant hepatitis B viral infection was found to present as larger tumors in younger patients with less severe liver dysfunction. Hepatocellular carcinoma with concomitant hepatitis C viral infection was often detected in follow-up studies when it was small.     

Frequent detection of hepatitis B virus X-gene DNA in hepatocellular carcinoma and adjacent liver tissue in hepatitis B surface antigen-negative patients

Hepatitis B virus is associated with human hepatocellular carcinoma. We performed polymerase chain reaction for the X, C, S, and preS2/S regions of the viral genome in 23 hepatitis B surface antigen-negative hepatocellular carcinomas and adjacent liver. Hepatitis B viral genomes were detected in 17 of 23 tumors and adjacent tissues (73.9%). Among recognized transactivators, the X gene was present in 16 (69.6%) cases of hepatocellular carcinoma, but preS2/S was detected in only 7 (30.4%). Hepatitis B virus C and S regions were detected in 3 (13.0%) and 9 (39.1%) hepatocellular carcinomas, respectively. Serologic study revealed antibodies to hepatitis B surface antigen, hepatitis B core antigen, and hepatitis B e antigen in 14 patients; among these, X-gene DNA was detected in 12 of 14 tumors (85.7%). The X gene was also detected in 4 of 9 tumors of seronegative patients. The X gene, present in many hepatocellular carcinomas, may promote hepatocellular carcinoma in hepatitis B surface antigen-negative patients.     

Preoperative chemoembolization of hepatocellular carcinoma: a comparative study

INTRODUCTION: Inappropriate discharges and/or improper inhibition of bradycardia pacing due to oversensing of extraneous signals in implantable cardioverter defibrillators (ICDs) have been described. With one exception, no previous report involving an intact lead system has cited myopotential oversensing as the cause. METHODS AND RESULTS: Two case reports of myopotential oversensing by a dual chamber ICD system are reported. In the first patient suffering from chronic pulmonary obstructive disease, intermittent myopotential sensing during labored respiration resulted in episodic inhibition of bradycardia pacing. In the second patient, oversensing of sustained myopotentials generated during strenuous isometric activity resulted in an inappropriate ICD discharge. For both, the ICD system consisted of a CPI model 1810 Ventak AV used in conjunction with a model 0125 Endotak lead, incorporating integrated bipolar sensing. CONCLUSION: Although modern ICDs have proven to be highly effective in detecting and terminating malignant tachyarrhythmias, the opportunity for improving their detection specificity remains.     

Significance of the minor i and t determinants of hepatitis B virus in hepatocellular carcinoma

Stored sera from asymptomatic hepatitis B virus (HBV) carriers and hepatitis B virus surface antigen-positive hepatocellular carcinoma (HCC) patients were tested for HBV subtypes, such as subtype determinants d, y, w, r and also antigenic determinants isoleucine (i) and threonine (t) by direct S gene nucleotide sequencing. Significant changes in minor i and t determinants in hepatocellular carcinoma patients with adr hepatitis B carriers were seen. The adr subtype with t determinant was present in 14/25 (56%) of HCC patients compared with only two of 28 (7%) in asymptomatic hepatitis B carriers (P<0.001). However, the adr subtype with i determinant was present in nine of 25 (36%) of the HCC patients and also present in 24/28 (86%) of asymptomatic carriers (P<0.001). No significant changes were seen with the adw subtypes. These results show that i and t minor determinant changes are more common with adr subtypes associated with HCC than with the adw subtype. Whether these subtle changes are pathologically relevant or only a polymorphism of hepatitis B genotypes will depend on subsequent follow-up studies.     

Variations of hepatitis B virus core gene sequence in Western patients with chronic hepatitis B virus infection

Heterogeneity of the hepatitis B virus (HBV) core gene has been reported to be associated with the presence of active liver disease in Japanese patients with chronic HBV infection. This study evaluated the significance of HBV core gene heterogeneity in Western patients with chronic HBV infection. The hepatitis B virus precore/core gene from 45 patients (inactive:active liver disease ratio 16:29) was amplified from serum by polymerase chain reaction (PCR). Gel electrophoresis was employed to detect large deletions. The PCR amplicons from 13 patients (all HBV serotype adw but with a different spectrum of liver disease) were cloned and sequenced. Hepatitis B surface antigen (HBsAg) serotypes were tested by enzyme immunoassay (EIA) and hepatic expression of HBV antigens was assessed by immunohistochemistry. The HBV core gene was amplified from the serum of all 45 patients. Three patients had mixed infection with both precore mutant and wild-type HBV and all three had active liver disease. No patient had a large deletion of the HBV core gene. Hepatitis B virus core gene sequence variations were more common in the midcore region and there was no difference in the number of silent and missense substitutions between those with inactive and active liver disease. There was no correlation between the nucleotide or encoded amino acid substitutions and the clinical and biochemical parameters, including the subsequent response to interferon-alpha therapy (n = 37) or hepatic HBV antigen expression. Variation of the HBV core gene was not found to be preferentially associated with active liver disease in Western patients with chronic HBV infection. The pattern of hepatitis B core gene variation is in accord with the genomic organization of HBV.     

Human hepatitis B virus and hepatocellular carcinoma. I. Experimental infection of tree shrews with hepatitis B virus

A cross-sectional study involving 771 children under the age of one year, was carried out in a traditional area of urban Ilorin, Nigeria, to determine how socio-economic conditions and feeding practices relate to diarrhoeal disease among infants. After adjustment has been made (through logistic regression) for covariates, five factors had significant association with diarrhoeal disease. These are the age of the child, parity, mother's education, availability of household kitchen and the feeding of semi-solid food to the infants. The lowest diarrhoeal rate occurred in infants aged 0-3 months while the highest rate occurred among infants seven to nine months old (Odds Ratio = 4.2). Children who were of the fifth or higher birth order had significantly higher risk of diarrhoea when compared with those who were of the first or second birth order (OR = 1.62; P < 0.05). Children of mothers with secondary education had significantly higher risk of diarrhoea compared with children of illiterates (OR = 1.9; P < 0.05). Households that had no kitchen had significantly higher risk of infantile diarrhoea than households with kitchen facilities (P < 0.01). Finally, infants receiving semi-solid food had higher risk of diarrhoea compared to those children not receiving semi-solid food (P < 0.05). Diarrhoeal disease awareness campaign to educate mothers on the dangers of childhood diarrhoea and how to prevent it, through proper hygiene, especially, food hygiene, is advocated.     

Past exposure to hepatitis B virus as a risk factor for hepatocellular carcinoma in patients with chronic liver disease

Maltitol is a disaccharide alcohol that is produced by hydrogenation of maltose and exhibits resistance to intestinal disaccharidases. We demonstrated previously that maltitol stimulated transepithelial diffusional calcium transfer in the ileum, accompanied by an elevation of intestinal calcium absorption as well as calcium retention in the body. In this study we examined whether the maltitol-induced increase in the diffusional transfer of intestinal calcium absorption leads to an alteration of the physical properties of bones in the weanling rats which exhibit the maximal level of intestinal active calcium absorption. Rat pups were removed from dams at 24 d of age and were fed the diets containing either maltose (control) or maltitol and a requisite amount of calcium (0.52%) for 21 d. Balance studies performed during the final 5-d period showed that maltitol-fed rats had greater calcium retention and calcium absorption. The breaking force of femoral bones was 13% greater in the rats fed the maltitol diet than in controls. The calcium content and dry weight of both femurs and tibias, as well as the bone mineral density of tibias, were elevated in the rats fed the maltitol diet. In a separate experiment, gastric intubation of maltitol-containing diet increased the serum calcium concentration in the portal vein at 2 and 4 h compared to controls. These results indicate that the maltitol-induced increase in the intestinal calcium absorption through paracellular pathway leads to enhancement of the calcium content and the breaking strength in the bone of weanling rats.     

Nucleotide sequence and phylogenetic analysis of Newcastle disease virus isolates from recent outbreaks in Taiwan

Portions of the hemagglutinin neuraminidase (HN) gene of Newcastle disease virus (NDV) isolates from two recent outbreaks were sequenced to investigate epidemiology of this disease in Taiwan. These NDV isolates were all viscerotropic velogenic according to the clinical lesions produced in chickens. Sequence data were obtained from 14 NDV isolates (12 from 1995 and 2 from 1984). All isolates differed in their nucleotide sequences (from 0.3 to 15.3%), and represented potentially different strains of NDV. Phylogenetic analysis revealed that these isolates are closely related to viruses isolated from Japan and Malaysia. Some viruses isolated in 1995 appeared to evolve from viruses isolated in 1984. The results suggest that the 1995 outbreak of Newcastle disease (ND) in Taiwan may have been caused by multiple strains of velogenic NDV that have cocirculated in Taiwan for some time. Moreover, NDV isolates from racing pigeons were very similar to isolates from chickens in the same period, suggesting that both domestic and free-living birds were involved in the spread of ND in Taiwan.     

The complete sequence of the genomic RNAs of spinach latent virus

We describe the sequence for the complete genome of spinach latent virus (SpLV). Comparisons of this genome with that of the only other complete genome described for a species within the genus Ilarvirus (citrus leaf rugose virus-CiLRV) indicate that while there are marked differences between the RNA 3 of the two viruses, their respective RNAs 1 and 2 share many similarities. However, the putative 2a protein of SpLV contains a C2H2 type "zinc finger"-like motif located towards the carboxy terminal of the protein which is absent in CiLRV and has not been reported for other members of the family Bromoviridae. A second open reading frame (2b), located at a similar position to that described for the cucumoviruses, occurs in the RNA 2 of both SpLV and CiLRV. The putative coat protein of SpLV is similar to that of citrus variegation virus (CVV) and asparagus virus 2 (AV-2), both members of subgroup 2 of the ilarviruses. We have subsequently demonstrated a serological relationship between SpLV and other viruses in subgroup 2 and suggest that SpLV should be included in this subgroup rather than remain in a separate group (subgroup 6). However, while the putative movement protein of SpLV is remarkably similar to that of AV-2, it shows little relationship with the corresponding protein of CVV and the lack of similarity suggests that a recombination event may have occurred in the past. The relationship between the genera Alfamovirus and Ilarvirus is discussed in the light of the data for the genome of SpLV and recently published information for other members of the genus Ilarvirus.     

Inhibition of hepatocellular carcinoma development in hepatitis B virus transfected mice by low dietary casein

In a comprehensive human ecological study, primary liver cancer has been shown to be highly significantly associated with 1) the prevalence of persistent infection with hepatitis B virus (HBV) and 2) plasma cholesterol concentrations that are, in turn, associated with the consumption of animal based foods. In rat studies, aflatoxin-induced hepatocellular carcinoma is substantially prevented by decreasing the intake of animal based protein (casein), a hypercholesterolemic nutrient. Thus the development of primary liver cancer associated with persistent HBV infection or with aflatoxin exposure may be controlled by reduced intake of animal-based proteins. Transgenic mice transfected with an HBV gene fragment containing the viral transactivator of hepatis B virus, HBx, which induces the formation of hepatocellular carcinoma, were used to examine the ability of dietary casein to modify tumor formation. Reducing the concentration of dietary casein to 6% from the traditional level of 22% markedly inhibited (by 75%) hepatic tumor formation in these transgenic mice. Tumor development also was substantially altered by interchanging dietary casein concentration well after tumor development had begun (at 8 months), increasing by 173% from the expected yield when casein intake was increased and decreasing by 99% when casein was reduced. These findings suggest that the development of liver tumor formation among individuals persistently infected with HBV may be controlled by minimizing or eliminating the intake of animal protein-based foods.     

Mapping of chromosomal imbalances in pancreatic carcinoma by comparative genomic hybridization

To identify recurrent chromosomal imbalances in pancreatic adenocarcinoma, 27 tumors were analyzed by using comparative genomic hybridization. In 23 cases chromosomal imbalances were found. Gains of chromosomal material were much more frequent than losses. The most common overrepresentations were observed on chromosomes 16p (eight cases), 20q (seven cases), 22q (six cases), and 17q (five cases) and under-representations on a subregion of chromosome 9p (eight cases). Distinct high-level amplifications were found on 1p32-p34, 6q24, 7q22, 12p13, and 22q. These data provide evidence for a number of new cytogenetically defined recurrent aberrations which are characteristic of pancreatic carcinoma. The overrepresented or underrepresented chromosomal regions represent candidate regions for potential oncogenes and tumor suppressor genes, respectively, possibly involved in pancreatic tumorigenesis.     

Hepatitis B virus deoxyribonucleic acid in kidney cells probably leading to viral pathogenesis among hepatitis B virus associated membranous nephropathy patients

Venous occlusion was used in 8 patients with liver cirrhosis and in 10 normals to investigate the pathomechanism of long-term elevation of plasma von Willebrand factor antigen (vWFAg) in liver cirrhosis. The following parameters were determined at baseline, and immediately, 60 min and 24 h after 10 min venous occlusion: vWFAg, ristocetin cofactor (RiCoF), in vitro platelet retention (Adeplat T), and tissue-type plasminogen activator (t-PA). Every baseline value in the liver cirrhosis group was significantly higher than in the controls. In both groups the 10-min values were significantly higher than their corresponding baseline results. Hence, comparing the two groups, in liver cirrhosis a significantly higher release of vWFAg and t-PA could be observed. These findings suggest on the one hand that the increased release contributes substantially to the sustained elevation of plasma vWF level in liver cirrhosis. On the other hand, the results indicate that not only the vascular surface of the diseased liver but most probably the total endothelium plays an important role in this phenomenon.     

Prevention of hepatitis B and C virus infection for prevention of cirrhosis and hepatocellular carcinoma

In Taiwan, cirrhosis and hepatocellular carcinoma (HCC) have been common in medical practice since the 1960s. In 1969, Taiwan was shown to be a hyperendemic area of hepatitis B virus (HBV) infection with a high rate of hepatitis B surface antigen (HBsAg) positivity, 19% of the population being infected before the fourth decade of life. There is evidence indicating that more than 80% of chronic hepatitis, cirrhosis and HCC are the sequelae of chronic HBV infection. In 1984, after 3 years of preparation, a programme to control cirrhosis and HCC began. All neonates born to HBsAg+ mothers were given Pasteur plasma-derived vaccine 5 micrograms i.m. at 1, 5 and 9 weeks with a booster at 12 months. In 1986, all neonates were included in this programme. In addition, beginning in 1987, all non-vaccinated preschool children were also immunized and susceptible medical personnel and people from HBsAg+ households were recommended to receive the vaccine. Using data obtained from the 7-year evaluation study on the efficacy of this vaccine and some historical data, the HBsAg positivity rate in people born in the first few years after 1986 was estimated to be 2.6%. This rate is expected to decrease to 0.2% in those born after around 1990. In July 1992, an anti-hepatitis C virus (HCV) test was included in blood donor screening tests. This was followed by a decrease in the incidence of post-transfusion hepatitis (PTH) from 13 to 2.5% and there have been no anti-HCV+ PTH cases since. However, without immunization, the prevalence of HBsAg decreased among children in Taipei in 1989. This coincided with the widespread use of disposable syringes and needles and an improvement in the sterilization of medical instruments. Therefore, it is likely that HCV infection may also decrease as a result of these practices. Through the use of immunization and improved medical procedures, chronic hepatitis, cirrhosis and HCC may decrease in Taiwan by around 95%.     

Sequential changes in full-length genomes of hepatitis B virus accompanying acute exacerbation of chronic hepatitis B

BACKGROUND/AIM: During the course of persistent hepatitis B virus infection, viral replication markedly decreases after acute exacerbation of liver inflammation accompanied by emergence of antihepatitis B e antibody (anti-HBe) and/or anti-hepatitis B surface antibody (anti-HBs). In some cases, however, persistent viral replication continues even after such exacerbation with or without HBeAg/anti-HBe seroconversion. The aim of the present study was to investigate the extent of genetic variations of HBV in this phenomenon. METHODS: Full-length HBV genomes were amplified by polymerase chain reaction from sera of three patients before and after acute exacerbation and were directly sequenced. RESULTS: In the whole genomes of 3215 nucleotides, only six nucleotide mutations for six amino acid substitutions (2 in the surface gene, 2 in the X gene, 1 in the core gene and 1 in the polymerase gene) were observed in patient 1, 15 mutations for 14 amino acid substitutions (1 in the pre-core codon 28, 4 in the surface gene, 4 in the core gene and 5 in the polymerase gene) were observed in patient 2, and 5 mutations for 6 amino acid substitutions (2 in the surface gene, 2 in the X gene, pre-core stop codon mutation and 1 in the polymerase gene) were observed in patient 3. Substitution in the a determinant of the surface gene, which encodes target epitopes for neutralizing antibodies, as well as those in the pre-core/core gene, which encodes epitopes for cytotoxic T cells, were mainly found. CONCLUSION: HBV that remained after the emergence of anti-HBe and anti-HBs are considered to possess mutations in epitopes for both humoral and cellular immunity. These mutant HBV may be involved in the pathogenesis of persistent hepatic injury after acute exacerbation.     

Detection and sequence analysis of hepatitis B virus integration in peripheral blood mononuclear cells

A PCR-based technique was used to detect hepatitis B virus (HBV) integration in peripheral blood mononuclear cells from patients with chronic hepatitis B. Integrated HBV DNA sequences, with virus-cell junctions located in the cohesive region between direct repeat 1 (DR1) and DR2, were found in 2 of 10 studied patients.     

Sensitive and reliable detection of genomic imbalances in human neuroblastomas using comparative genomic hybridisation analysis

Deletions of the short arm of chromosome 1, extra copies of chromosome 17q and MYCN amplification are the most frequently encountered genetic changes in neuroblastomas. Standard techniques for detection of one or more of these genetic changes are karyotyping, FISH analysis and LOH analysis by Southern blot or PCR. Each of these techniques has its own particular limitations. More recently, comparative genomic hybridisation (CGH) was introduced for detection of genomic imbalances including deletions, duplications and gene amplification. We evaluated the sensitivity and reliability of CGH for detection of the most frequently encountered genetic changes in neuroblastoma. For this purpose a panel of well-characterised neuroblastoma cell lines as well as a series of 11 primary neuroblastomas was analysed. Our results show that CGH is a valuable tool for the genetic characterisation of neuroblastomas, both for the detection of frequently occurring genomic imbalances and for the identification of previously unnoticed genetic changes.