Long-term clinical impact of occult hepatitis B virus infection in chronic hepatitis B patients.

BACKGROUND/AIMS: Long-term clinical outcomes of occult hepatitis B virus (HBV) infection were studied. METHODS: Fifteen chronic hepatitis B patients were monitored for a median of 4.4 years (range 0.9-15.3) after hepatitis B surface antigen (HBsAg) seroclearance. Serum HBV DNA was measured by real-time detection polymerase chain reaction. Thirteen patients underwent liver biopsies at the end of follow-up and liver histology was evaluated by Ishak score. Liver HBV DNA was also measured for 12 patients. RESULTS: At the end of follow-up, HBV viremia was absent in 13 (87%) patients, and antibody titers to hepatitis B core antigen showed an inverse correlation with time from HBsAg seroclearance (r=-0.554; P=0.0040). However, all patients retained liver HBV DNA and tested positive for the covalently closed circular HBV DNA replicative intermediate. The hepatic HBV DNA loads had no relation to liver histology. Paired biopsies from 11 patients disclosed that each necroinflammatory score significantly improved after HBsAg seroclearance. Amelioration of liver fibrosis was also evident in eight (73%) patients (P=0.0391 by signed rank test). CONCLUSIONS: A long-standing but strongly suppressed HBV infection may confer histological amelioration after HBsAg seroclearance.

Effect of maternal separation on feeding behavior of rats in later life.

Effects of maternal separation on feeding behavior, particularly on rebound hyperphagia, in adult rats were examined. Time-restricted scheduled feeding (2 h per day for 6 days), was given at the age of 3, 6, 9 or 12 weeks in rats that were maternal separated from postnatal days (PD) 1-21 and control rats. Following the time-restricted scheduled feeding, rats were fed freely for 24 h (rebound hyperphagia). Body weight, daily normal food consumption and food consumption during time-restricted scheduled feeding and rebound hyperphagia were measured. Body weight of 3-week-old maternally separated rats were less than those of control rats. There was no significant difference in normal daily food consumption. Food consumption during rebound hyperphagia was significantly increased in 6- to 9-week-old female maternally separated rats, but there was no difference observed in males. Postnatal maternal separation enhanced rebound hyperphagia of female rats in later life. These results indicate that postnatal maternal separation made rats more vulnerable to the development of abnormal feeding behavior in response to food restriction in later life.

[Development of the quantification method of TTV-DNA and clinical application].

We developed a method to measure the quantity of TTV-DNA. This measurement is based on the principle of the real time PCR method using the TaqMan probe. By measuring the change of the fluorescent intensity caused by FRET, we could detect the amount of TTV-DNA. This method has the characteristics that the possibility of the contamination is very rare when it is compared with the usual PCR method, because the reaction system contains UNG and dUTP. This quantification method is useful for the future research of TTV to study the relationship between this virus and diseases.

[Detection rate of TTV-DNA in healthy medical workers].

We examined the positive rate in healthy medical workers about TT virus (TTV) which was a new hepatitis virus reported in 1997. The healthy medical workers showed a positive rate of 24%. Because there was no significant difference of the positive rate between the medical workers and general healthy persons we could not conclude that the positive rate was influenced by their profession. Generally it is known that a positive rate changes according to PCR primers used for the measurement. As for TTV as well, it is reported that a positive rate is greatly dependent on the selected region of primer. Therefore, we must pay attention to the evaluation of the positive rate for each assay system, especially using different primers.

Telomerase reverse transcriptase mRNA expression and telomerase activity in hepatocellular carcinoma.

Human telomerase reverse transcriptase (hTERT) has been identified as the catalytic subunit of human telomerase. To clarify the clinical significance of hTERT mRNA in hepatocellular carcinoma (HCC), we investigated the relationship between telomerase activity and hTERT mRNA in human HCC and non-HCC tissues. The hTERT mRNA was detected in 17 (89.47%) of 19 livers with HCC and in 4 (21.05%) of 19 noncancerous tissues from these livers. Telomerase activity was detected in 17 of the 19 tumor tissues (89.47%) and in 4 of the 19 nontumor tissues (21.05%). The hTERT mRNA was detected in all tissues that were telomerase-positive and it was undetected in all tissues that were telomerase-negative. The correlation between the expression of hTERT mRNA and human telomerase activity in this study indicates that hTERT mRNA could be useful to diagnose cancer. Also, as telomerase production may be under the control of hTERT mRNA, the possibility is great that noncancerous liver tissue with chronic liver diseases acquires HCC when the hTERT mRNA is positive.

Levels of telomerase catalytic subunit mRNA as a predictor of potential malignancy.

This study investigated the relationship between the levels of human telomerase reverse transcriptase (hTERT) mRNA and that of telomerase activity in hepatocellular carcinoma (HCC). A significant correlation between hTERT mRNA expression and telomerase activity by transfecting the gene encoding hTERT into telomerase-negative human fibroblast cells has clearly been demonstrated. However, the relationship between levels of telomerase activity and that of hTERT mRNA has yet to be elucidated. In this study, the levels of hTERT mRNA were analyzed in 24 HCC patients by real-time PCR. And the intensity of telomerase activity was analyzed by fluorescence-based TRAP method. The difference of hTERT mRNA level was highly significant between tumor tissues and non-cancerous liver tissues. And there were significant correlations between the levels of hTERT mRNA and that of telomerase activity (r=0.751) in tumor tissues. We observed a strong correlation between levels of hTERT mRNA and that of telomerase activity in HCC. Our results suggest that the levels of hTERT mRNA would be useful in genetic diagnostic tests, instead of telomerase activity, to screen at-risk patients of HCC in human liver tissues.

Enhanced hepatocyte growth factor expression associated with prolonged rat hepatic allograft survival in recipients pretreated with donor-specific blood.

BACKGROUND: Pretransplantation injection of freshly heparinized donor blood (donor-specific blood transfusion, or DST) significantly prolongs the survival of hepatic allografts from ACI(RT1a) to LEW(RT1l) rats. We investigated hepatocyte growth factor (HGF) expression in rat hepatic allografts of recipients pretreated with or without DST. METHODS: The levels of HGF mRNA and protein in hepatic allografts were determined after transplantation. The localization of HGF+ cells was identified with a rat anti-HGF monoclonal antibody. RESULTS: Plasma HGF concentrations in transplanted rats treated with DST were significantly and persistently increased compared to untreated rats with hepatic allografts. The number of HGF+ cells in hepatic allografts of recipients pretreated with DST on day 14 was significantly greater than that in allografts of untreated recipients on day 7. HGF+ cells were also found in the marginal zone and red pulp of recipient spleens. Northern blot analysis revealed the presence of three HGF+ cell phenotypes: HGF+ED1+, HGF+ED2+, and HGF+ED1-ED2-. Most HGF+ cells were ED1-ED2-. In situ hybridization demonstrated HGF mRNA in the mononuclear cells in the portal and sinusoidal areas as well as the marginal zone and red pulp in both DST-treated and untreated recipient spleens. CONCLUSIONS: Enhanced HGF expression in rat hepatic allografts is associated with immunologic unresponsiveness induced by DST.

Phenotypic conversion from t(8;21) acute myeloid leukemia to MLL gene rearrangement-positive acute lymphoblastic leukemia.

Phenotypic conversion from acute myeloid leukemia (AML) to acute lymphoblastic leukemia (ALL) is rare. A 38-year-old man was initially diagnosed as having AML (FAB-M2) associated with the t(8;21)(q22;q22) chromosomal abnormality. The blasts showed myeloperoxidase (MPO) activity and CD13 antigen expression. He showed complete remission after standard chemotherapy for AML. However, the patient relapsed with blasts showing ALL morphology (FAB-L1), MPO negativity, and CD19 antigen expression 33 months after cessation of AML therapy. Cytogenetic analysis at relapse was unsuccessful. Molecular analysis of ALL blasts revealed immunoglobulin heavy-chain gene and MLL gene rearrangements but no AML1 gene. MLL gene rearrangement or the 11q23 chromosomal abnormality has been associated with therapy-related leukemia. The subsequent ALL in our patient may have been induced by the chemotherapy including daunorubicin, known as a topoisomerase II inhibitor.

[Extramedullary relapse in the external auditory canal in a patient with acute promyelocytic leukemia treated with all-trans retinoic acid and autologous peripheral blood stem cell transplantation].

A 41-year-old man was given a diagnosis with of acute promyelocytic leukemia (APL) in August 1994. A chromosome analysis showed 46, XY, t(15; 17) and 47, XY, idem, +8 at that time. Because initial induction chemotherapy (BHAC-DMP) has not been successful, the patient was given 45 mg/m2 of all-trans retinoic acid (ATRA) and achieved complete remission (CR) after 26 days on this regimen. Following intensified chemotherapy, he received an autologous peripheral blood stem cell transplant (PBSCT) with high-dose busulfan and cyclophosphamide in April 1995. Competitive RT-PCR for PML-RAR alpha mRNA did not find any of APL cells in the collected stem-cell fraction. Although the patient remained in CR without therapy, a myeloblastoma was found in his left external auditory canal in August 1996. Recurrence in bone marrow, moreover, was discovered the following month. A chromosome analysis of bone marrow cells showed 47, XY, t(15; 17), +8 at this time. Thus, the extramedullary relapse developed after autologous PBSCT. This case provides information linking ATRA to the development of extramedullary relapse in patients with APL.

Genetic diagnostic test of hepatocellular carcinoma by telomerase catalytic subunit mRNA.

This study investigated the relationship between telomerase activity and telomere length and between telomerase reverse transcriptase (hTERT) mRNA and telomere length. Both cancerous and non-cancerous tissues were studied in individuals with hepatic carcinoma. In this study, the telomere length in HCC livers had a wide range, no clear significant correlation was found between hTERT mRNA and telomere length. Telomerase activity was more strongly correlated with hTERT mRNA than with telomere length. The correlation between hTERT mRNA and telomerase activity shown here indicates that hTERT mRNA has potential for cancer diagnosis.

Alteration of the CDKN2A gene in pancreatic cancers: Is it a late event in the progression of pancreatic cancer?

Various genetic changes are involved in progression of various cancers. We examined alterations (deletion, sequence abnormalities, methylation) of the CDKN2A gene in cell lines and tumor tissues of pancreatic cancers. Some alterations of this gene were found in all the 12 cell lines examined. In the primary lesions of pancreatic cancers, homozygous or hemizygous deletion were found in 8 of 24 ductal carcinoma and 4 of 9 other types of carcinomas. It appears that there is an association between the alteration of this gene and tumor size, regional lymph node metastasis and hematogenous distant metastasis in the ductal carcinoma, but not in the other types of carcinomas. All the 5 liver metastatic lesions of the ductal carcinoma examined revealed homozygous or hemizygous deletion and 3 bp deletion. These results suggest that inactivation of the CDKN2A gene occurs more frequently in cell lines than in pancreatic cancer tissues. Such genetic events on the CDKN2A gene may play an important role possibly at a later step in the progression of pancreatic ductal carcinoma.

A germline mutation abolishing the original stop codon of the human adenine phosphoribosyltransferase (APRT) gene leads to complete loss of the enzyme protein.

Adenine phosphoribosyltransferase (APRT) is a purine metabolic enzyme and a homozygous deficiency in this enzyme causes 2,8-dihydroxyadenine urolithiasis. Various germline abnormalities have been described, but we report here a unique type of germline mutation in a homozygous individual (SY) who had excreted 2,8-dihydroxyadenine crystals. In SY, TCA was substituted for the physiological stop codon TGA. This base substitution generates a new HinfI restriction site, and, using the polymerase chain reaction and subsequent digestion by this enzyme, it was confirmed that SY is homozygous for the base substitution. This base change is unique in that it generates an open reading frame that extends to the poly(A) addition site. The amount of mRNA in transformed B cells from SY was approximately a quarter of that in control subjects and no APRT proteins were detected. In eukaryotes, unlike in prokaryotes, no rescue systems for defective polypeptide termination caused by a missing stop codon have been found. Therefore, the outcome of the defect of SY is unclear from present knowledge about termination of polypeptide synthesis. Investigations into the mechanisms of the absence of protein in the cells of SY may lead to a better understanding of the physiological and nonphysiological termination of polypeptide synthesis in eukaryotic cells.

Prevalence and molecular epidemiology of GB virus C/hepatitis G virus infection in Mongolia.

We studied the prevalence of GB virus C/hepatitis G virus (GBV-C/HGV) infection among 112 patients with liver disease and 121 blood donors in Ulaanbaatar, Mongolia. Reverse transcription and polymerase chain reaction were employed to detect GBV-C/HGV RNA using the specific primers derived from the 5'-untranslated region (5'-UTR) of the GBV-C/HGV genome. Nucleotide sequences of all positive samples for GBV-C/HGV RNA were determined. The sequences were analyzed by a molecular evolutionary method. Twenty-five (10.7%) of 233 people were positive for GBV-C/HGV RNA. Eight (6.6%), 11 (9.1%), and 30 (24.8%) blood donors were positive for GBV-C/HGV RNA, HBsAg, and anti-HCV, respectively, although 17 (15.2%), 65 (58.0%), and 64 (54.5%) patients with liver disease were positive for each viral marker. The prevalences of GBV-C/HGV RNA, HBV, and HCV in the patients were significantly higher than those in blood donors (P < 0.05). There was no significant difference in the prevalence of anti-HCV among people with and without GBV-C/HGV RNA, while the prevalence of HBsAg among people with GBV-C/HGV RNA was significantly higher than among those without GBV-C/HGV RNA (P < 0.05). The molecular evolutionary tree showed that GBV-C/HGV was a heterogeneous virus and all strains could be divided into 2 types. One is the same phylogenetic type as HGV, and the other is a new type that is different from GBV-C and HGV.

Comparison of the nucleic acids of helical and coccoid forms of Helicobacter pylori.

The nucleic acids of the helical and coccoid forms of Helicobacter pylori were studied to determine if the coccoid forms are "viable (capable of growing) but nonculturable." Using a reference strain (NCTC 11638) and five clinical strains, the nucleic acid contents, DNA integrity, and results of PCR and reverse transcription-PCR (RT-PCR) were compared for helical H. pylori and coccoid forms induced using glycochenodeoxycholic acid or bismuth citrate. The DNA and RNA contents of the coccoid forms were respectively 6.8- and 8.1-fold lower than those of helical H. pylori after 3 days of induction and 11.5- and 14.7-fold lower after 7 days. Agarose gel electrophoresis of DNA extracted from the coccoid forms after 3 days of induction showed a smear pattern indicating DNA cleavage, whereas DNA from helical H. pylori showed a single band with a high molecular mass. After 12 days of induction, all RNA samples from 100% coccoid cultures were negative for the mRNA of urease A or the 26-kDa species-specific protein by RT-PCR. However, most RNA samples obtained after 3 or 7 days of induction were positive at low levels despite the lack of recovery from these cultures. These results suggest that the coccoid form of H. pylori has impaired genomic DNA and is in the process of cellular degeneration, thus being still alive but nonincreasable.

Three different GB virus C/hepatitis G virus genotypes. Phylogenetic analysis and a genotyping assay based on restriction fragment length polymorphism.

The 5'-untranslated region (5'-UTR) sequences of 33 GB virus C/hepatitis G virus (GBV-C/HGV) obtained from different geographic areas were determined through reverse-transcription polymerase chain reaction and dideoxy chain termination sequencing, the alignment of sequences, the estimation of the number of nucleotide substitution per site, and construction of phylogenetic trees. The 5'-UTR of GBV-HGV was found to be heterogeneous, with 70.9-99.5% homology. Three distinct phylogenetic branches were observed consistently in all phylogenetic trees. GBV-C is the prototype for one, HGV for another, and there is a new branch which consisted of GBV-C/HGV isolates from Asia. Genotype-specific restriction sites for the restriction enzymes, ScrFI and BsmFI, were identified, and a simple restriction fragment polymorphism analysis was developed for genotyping. These data provide evidence that GBV-C/HGV consists of three different genotypes. Our simple genotyping assay will also provide a tool for epidemiological studies of GBV-C/HGV infection.

GB virus C/hepatitis G virus infection in southern China.

The prevalence of GB virus C/hepatitis G virus (GBV-C/HGV) infection among intravenous drug users (IVDUs), patients with liver diseases, and blood donors in Nanning, southern China was studied. GBV-C/HGV RNA was detected by reverse transcription polymerase chain reaction with primers derived from the 5'-untranslated region. GBV-C/HGV RNA was detected in 64 of 85 IVDUs, 20 of 80 persons with liver disease, and 1 of 50 blood donors. Among IVDUs, GBV-C/HGV infection was associated with antibodies to hepatis C virus (HCV) and with hepatitis B surface antigen (HBsAg). Eleven nucleotide sequences were determined and analyzed by molecular evolutionary analysis. In a phylogenetic tree, the isolates were grouped in three clusters with GBV-C and HGV grouped in two clusters. These data indicate that GBV-C/HGV infection is common in China among IVDUs but uncommon among persons with liver disease without HBsAg or anti-HCV and that there is a new group of GBV-C/HGV.

[The detection of minimal residual disease by DEK/CAN chimeric m-RNA in a case of AML M2 with translocation t(6;9) (p23;q34) after chemotherapy and peripheral blood stem cell transplantation].

A 18-year old female with acute myelogenous leukemia (AML), M2 had translocation: t(6;9) (p23; q34). The patient entered into hematological complete remission after two courses of BHAC-DMP chemotherapy with disappearance of cytogenetic abnormality. However, minimal residual disease (MRD) detected with DEK/CAN chimeric m-RNA by reverse transcription polymerase chain reaction (RT-PCR) was continuously observed, although decreased quantitatively, following several courses of consolidation and intensification chemotherapies. MRD was detected also in the harvested peripheral blood stem cells (PBSC). Leukemia relapsed with the reappearance of t(6;9) 2 months after the subsequent peripheral blood stem cell transplantation (PBSCT). Leukemia became refractory to chemotherapy, and the patient died 5 months thereafter.

Genotype of GB virus C/hepatitis G virus by molecular evolutionary analysis.

GB virus C/hepatitis G virus is a newly described virus. Classification of GB virus C/hepatitis G virus into genotypes has not been established. We analyzed nucleotide sequences within the 5' untranslated region of GB virus C/hepatitis G virus isolates and segregated these isolates into genotypes. Twenty serum samples with GB virus C/hepatitis G virus RNA from Australia, Cameroon, the Congo, Japan, Mongolia, and Bangladesh were studied. Reverse transcription and polymerase chain reaction were used to obtain GB virus C/hepatitis G virus RNA. After nucleotide sequences from the 5' untranslated region were determined, 68 nucleotide sequences, including 48 previously reported sequences, were analyzed by molecular evolutionary methods. The phylogenetic tree of the 5' untranslated region showed that all strains could be divided into three major genotypes, GB type (type 1), HG type (type 2), and Asian type (type 3). Bootstrap analysis indicated that the strains could be divided into three major genotypes but could not be further subdivided. Moreover, frequency histograms of pairwise distances between nucleotide sequences demonstrated only one peak. These result indicated that GB virus C/hepatitis G virus can be classified into three major genotypes, GB type (type 1), HG type (type 2), and Asian type (type 3), and should not be divided into minor subtypes.

GB virus C/hepatitis G virus infection among Japanese patients with chronic liver diseases and blood donors.

Recently, a novel hepatitis virus, GB virus C/hepatitis G virus (GBV-C/HGV), has been isolated. To elucidate the seroprevalence of chronic GBV-C/HGV infection in Japan and the phylogenetic relationship between Japanese strains and the strains previously reported, serum GBV-C/HGV RNA was detected by reverse transcription polymerase chain reaction (RT-PCR) in 203 patients with chronic liver diseases and 200 samples of voluntary blood donors. RT-PCR was performed with primers derived from the 5'-untranslated region which were conserved between GBV-C and HGV and distant from other flaviviruses including hepatitis C virus (HCV). The nucleotide sequences were determined by the dideoxy chain termination method. The phylogenetic analysis was performed by the neighbor-joining method. In 10 (4.7%) of 203 patients with chronic liver diseases and in 1 (0.5%) of 200 blood donor samples, serum GBV-C/HGV RNA was detected. Of 10 patients, 9 patients were positive for anti-HCV and negative for HBsAg, and 1 patient was positive for HBsAg and negative for anti-HCV. The phylogenetic analysis indicated that there were three major groups which were group 1 (GBV-C), group 2 (HGV), and group 3 (a group of Japanese strains). These data indicated that (1) there was a low prevalence of GBV-C/HGV infection in Japanese patients with chronic liver diseases, (2) a high proportion of patients with GBV-C/HGV infection had chronic HCV infection however, and (3) there were at least three groups in strains of GBV-C/HGV.