Search PubMed⌕ Search

Biomedical subjects

N Enomoto

Publications and source records attributed to N Enomoto.

At least 127 records · Page 7Linked to original sources
PubMed1994

Sequential change of the hypervariable region of the hepatitis C virus genome in acute infection.

Hepatitis C virus (HCV) infection is characterized by persistence of liver inflammation that often leads to end-stage liver disease, although the mechanisms are not fully understood. A hypervariable region (HVR) has been reported in the E2/NS1 region of the HCV genome, in which striking diversity is found among different HCV isolates. To investigate the association of the HVR alterations with the clinical courses of HCV infection, a longitudinal analysis of the HVR in patients with acute HCV infection was carried out. Plasma samples were obtained at several times in three patients with acute hepatitis C. Plasma RNA was extracted and reverse transcribed, and DNA fragments that included the HVR were amplified by PCR. The sequences of the HVR were directly determined from the PCR products by the dideoxy chain termination method, from which amino acid sequences were deduced. In all cases, plasma HCV-RNA disappeared with the improvement of the initial alanine aminotransferase (ALT) elevation, but HCV-RNA reappeared about 1 year later with or without deterioration of the hepatitis. In a case of sporadic acute hepatitis, the HCV in the recurrent phase had seven amino acid substitutions in the HVR compared with that in the acute phase, although no amino acid changes were noted during the initial acute phase. In a case of posttransfusion hepatitis, a marked difference was observed between the acute and the recurrent phases, with an amino acid homology of 30% (8/27). The mutation rate of the HVR had a tendency to accelerate as the HCV infection progressed to the chronic stage.(ABSTRACT TRUNCATED AT 250 WORDS)

Acute Disease↗
PubMed1994

Hepatitis C virus after interferon treatment has the variation in the hypervariable region of envelope 2 gene.

There is a hypervariable region in the envelope 2 gene of the hepatitis C virus genome, whose heterogeneity in different hepatitis C virus isolates has been suggested to be a result of the immune selection of escape variants. To determine the role of hypervariable region variants in the mechanism of resistance to interferon observed in 75-80% of interferon-treated patients with chronic hepatitis C, hypervariable region sequences were compared before and after interferon treatment. Eight patients with chronic hepatitis C were treated with recombinant interferon-alpha-2b. DNA containing the hypervariable region was obtained by reverse transcription-polymerase chain reaction from serial plasma samples of each patient and directly sequenced without cloning to determine changes in the predominant sequence. In two patients, hepatitis C virus-RNA was eliminated by interferon treatment. In the remaining six patients, hepatitis C virus-RNA was not eradicated. The predominant hepatitis C virus which survived interferon treatment was the mutant hepatitis C virus with 3-19 out of 81 nucleotide substitutions in the hypervariable region, resulting in 2-14 out of 27 amino acid changes. Most of the nucleotide substitutions were nonsynonymous, indicating that there were positive selections for amino acid changes in the hypervariable region. The change rate was significantly higher in patients whose plasma hepatitis C virus-RNA was consistently detectable during and after interferon treatment than in patients whose plasma hepatitis C virus-RNA became undetectable during treatment and reappeared after the cessation of the treatment (4.23 +/- 0.43 vs 0.77 +/- 0.20 x 10(-1)/site/year, p < 0.01). This suggests that the evolution of the hypervariable region was associated with the effect of interferon treatment. These results suggest that hypervariable region variants play an important role in maintaining persistent infection during interferon treatment by evading host immune surveillance.

Adult↗
PubMed1994

Detection and quantification of hepatitis C virus RNA replication in the liver.

To investigate the correlation between the replication of hepatitis C virus in liver and the clinical and histopathological features, we detected and quantified plus and minus strands of HCV-RNA in plasma and in livers of patients with chronic hepatitis C by a quantitative polymerase chain reaction. RNA was extracted from the plasma and liver tissue of ten patients with biopsy-proven chronic hepatitis C. The plus and minus strands of HCV-RNA were detected by a strand-specific reverse transcription with either sense or anti-sense oligonucleotide primers deduced from the hepatitis C virus genome, and a standard HCV-RNA with an enzyme restriction site was used to quantify the amount of HCV-RNA. Both plus and minus strands of HCV-RNA were detected from the liver tissue of all patients included. The amount of plus-stranded HCV-RNA in the liver was 10 times higher than that of minus-stranded HCV-RNA. Plus-stranded HCV-RNA was detected in the plasma in all patients, while the minus strand was not detected in any patient. There was a weak correlation between the amount of both strands of HCV-RNA in the liver and that of the plus strand in plasma. There was no significant correlation between the amount of liver HCV-RNA and serum alanine transaminase and aspartate transaminase levels, or histopathological findings in the liver. The present method of detecting and quantifying liver HCV-RNA is simple and sensitive; it may be used to detect residual hepatitis C virus replication after the disappearance of plasma HCV-RNA in acute hepatitis or in chronic hepatitis after interferon treatment.

Adult↗
PubMed1994

Fluctuation of hepatitis C virus quasispecies in persistent infection and interferon treatment revealed by single-strand conformation polymorphism analysis.

Hepatitis C virus (HCV) populations in vivo consist of heterogeneous mixtures of genetically different but closely related variants defined as a 'quasispecies'. The longitudinal fluctuation of HCV quasispecies populations in chronic hepatitis C has not been elucidated. Serial plasma samples were obtained from four patients with chronic hepatitis C (two patients without any treatment and two patients treated with interferon), and cDNA fragments containing the 5'-terminal region of the E2 gene of HCV were amplified from plasma RNA using PCR. Since conventional cloning of PCR products detects only a small part of the entire population, PCR products of each sample were separated by electrophoresis using single-strand conformation polymorphism (SSCP) analysis, which can distinguish DNA fragments of the same size as different electrophoretic bands depending on their sequence-specific conformation. Separated DNA fragments were recovered from SSCP bands in gels and their nucleotide sequences determined. SSCP electrophoresis separated PCR products into bands with different mobility. Sequence analysis of these bands confirmed that HCV populations in each patient are composed of quasispecies with different E2-hypervariable regions (HVR), which are known to contain antibody epitopes. Different patterns of variation in the HVR of quasispecies were observed in individual patients with different clinical features over time during chronic infection. Following interferon treatment, some quasispecies disappeared during the treatment and reappeared after the end of the treatment, whereas other quasispecies in the same patient remained during the treatment suggesting that the sensitivity to interferon is different among quasispecies.

Base Sequence↗
PubMed1994

[Fluctuation of HCV quasi-species population during interferon therapy; analysis by single strand conformation polymorphism].

We investigated the fluctuation of hepatitis C virus (HCV) quasispecies during interferon therapy by single strand conformation polymorphism (SSCP) analysis. In 13 of 16 interferon ineffective patients, the predominant HCV population was replaced with other quasispecies during the treatment. Especially, in 9 patients, a part of the HCV quasispecies, pre-existing before interferon therapy, became predominant after the therapy. These results indicate that sensitivity to interferon differs among HCV quasispecies and that interferon selects resistant HCV strains. Existence of such HCV quasispecies seems to be associated with interferon treatment failure.

Base Sequence↗
PubMed1993

Expression of the hepatocyte growth factor receptor in the regenerating rat liver.

The c-met oncogene product is a cell-surface receptor, which ligand is believed to be the hepatocyte growth factor. We studied the expression of c-met oncogene in the regenerating rat liver after either partial hepatectomy or CCl4-induced liver injury. Northern blot analysis showed that after partial hepatectomy the transcripts of c-met decreased at 8 h, reached the minimum at 36 h, and returned to the steady level on the seventh day. In contrast with the hepatectomized liver, the transcripts of c-met increased after CCl4 treatment. These observations suggest that c-met transcription may be regulated differently depending on regeneration signals.

Amino Acid Sequence↗
PubMed1993

Prevalence of hepatitis C virus infection among long-term hemodialysis patients: detection of hepatitis C virus RNA in plasma.

A high prevalence of anti-C100-3 antibodies has been reported in chronic hemodialysis patients. It is, however, unclear whether the results reflect true hepatitis C virus (HCV) infection in these patients, since false-positive results have been reported with this antibody assay. In the present study, plasma from 184 hemodialysis patients (110 males, 74 females) was examined for HCV-RNA by a reverse transcription-polymerase chain reaction method. Anti-C100-3 antibodies, second generation HCV antibodies, and anti-GOR antibodies were also examined by the respective EIAs. The positive rate of anti-C100-3 in the hemodialysis patients was 10.7% (20/184), which was significantly higher than the reported findings in the general population (P < 0.01). Using a second generation HCV antibody assay, the positive rate increased to 22%. HCV-RNA was detected in 15 of 184 patients (8.2%); 5 of 20 C100-3-positive patients (25%), and 10 of 164 C100-3-negative patients (6.1%). Serum alanine aminotransferase and gamma globulin levels were significantly higher in the patients with these HCV markers than those that were negative, while the history of blood transfusion was not related to the rate of the HCV markers. It is concluded that some hemodialysis patients have latent HCV infections that cannot be detected by currently available HCV antibody assays or routine biochemical liver function tests, and that the routes of transmission are not solely through blood transfusion.

Adolescent↗
PubMed1993

Correlation of plasma hepatitis C virus RNA levels with serum alanine aminotransferase in non-A, non-B chronic liver disease.

The relationship between plasma hepatitis C virus (HCV) RNA levels, antibody positivity, and hepatocellular damage were studied in 41 patients with non-A, non-B chronic liver disease. The patients were placed into two groups according to the plasma levels of HCV-RNA: plasma HCV-RNA level was estimated as high when detected by a one stage polymerase chain reaction (PCR) and as low when detectable only after a two stage PCR. Anti-HCV (first and second generation assays) and anti-GOR were also measured. The mean alanine aminotransferase (ALT) level of the high HCV-RNA group was 115 +/- 62 IU/l, whereas that of the low HCV-RNA group was 59 +/- 37 IU/l (P < 0.05). Patients with ALT levels above 100 IU/l had invariably a high level of HCV-RNA. There were no differences in clinical features in relation to the presence of anti-GOR or anti-HCV. Circulating HCV-RNA levels but not anti-HCV or anti-GOR antibodies correlated with hepatocellular damage.

Aged↗
PubMed1993

Detection of hepatitis C viral RNA in sporadic acute non-A, non-B hepatitis by polymerase chain reaction. Its usefulness for the early diagnosis of seronegative infection.

To determine the prevalence of hepatitis C viral infection in patients with sporadic non-A, non-B (NANB) acute hepatitis, hepatitis C viral RNA was studied in the plasma of 15 patients by reverse transcription-polymerase chain reaction assay. Plasma samples were sequentially obtained from 15 patients, and polymerase chain reaction was performed with two nested pairs of primers deduced from the 5'-non-coding region of hepatitis C viral sequences. Anti-C100 and anti-GOR antibodies were also measured with an enzyme-linked immunosorbent assay system. Plasma hepatitis C viral RNA was detected transiently in 7 of 15 patients (47%) at an early phase of the clinical course, while anti-C100 antibodies were detectable in only 2 (29%) of hepatitis C viral RNA-positive patients, and in 1 (13%) of the negative patients. Of 7 patients that were positive for plasma hepatitis C viral RNA, 4 (57%) had relapsing or protracted courses. In contrast, in all patients with undetectable hepatitis C viral RNA, hepatitis C viral RNA recovered and remained normal for at least 1 year. Thus, hepatitis C viral infection represents almost half the patients with acute sporadic NANB hepatitis, and detection of hepatitis C viral RNA in an early clinical phase is superior to anti-C100 measurement for diagnosing acute sporadic hepatitis C viral infection.

Adult↗
PubMed1993

Detection of hepatitis C virus RNA in the liver by in situ hybridization.

To examine HCV infection histologically, we attempted non-radioactive in situ hybridization of HCV-RNA in the liver. We amplified cDNA probe (360 base pairs) by PCR using the primers deduced from the core region of the HCV genome. The probe was labelled with digoxigenin by PCR and used for in situ hybridization on paraformaldehyde-fixed frozen liver sections. The hybrids were visualized immunohistochemically with alkaline-phosphatase-conjugated anti-digoxigenin and alkaline-phosphatase substrates. HCV-RNA-cDNA hybrids were detected in 21 of 24 patients with positive serum HCV markers, whereas there were no positive signals in the liver of 12 cases without HCV infection. The signal intensity of HCV-RNA-cDNA hybrids was abolished after RNase treatment. Various other specificity experiments also verified specific hybridization of HCV-RNA-cDNA. HCV-RNA was visualized in liver cells and most of them were regarded as hepatocytes from their characteristic features. The infected hepatocytes were frequently associated with mononuclear cell infiltration. Hepatocytes positive for HCV-RNA were sometimes binuclear and distributed in various patterns among cases tested. The present in situ hybridization of HCV RNA is highly sensitive and specific and the results suggest the host immune response to HCV-infected cells.

Hepacivirus↗
PubMed1993

Rapid sequence variation of the hypervariable region of hepatitis C virus during the course of chronic infection.

To evaluate the clinical significance of sequence variations in the hypervariable region of hepatitis C virus during the course of chronic infection, we performed pairwise comparison of the predominant nucleotide sequences. Hepatitis C virus RNA was extracted from two plasma samples obtained from 12 chronically infected Japanese patients over approximately 1 yr. Complementary DNA containing the hypervariable region was amplified by means of reverse transcription-polymerase chain reaction and was directly sequenced for determination of predominant sequences. In all 12 individuals, the predominant sequence of the hypervariable region at the second time point differed from that at the first time point, and significant sequence variation was observed during this short period. A high proportion of these nucleotide substitutions (90%) resulted in changes of predicted amino acid sequences, indirect evidence of in vivo positive selection for these variants. There appeared to be an important difference in the rate of nucleotide sequence variation of the hypervariable region between four patients with flare-ups of their ALT levels (1.54 to 2.24 x 10(-1)/genome site/yr) and eight patients with quiescent courses (0.13 to 1.21 x 10(-1)/genome site/yr). These results demonstrate that rapid sequence variations of the hypervariable region of predominant virus population take place during the natural course of chronic hepatitis C virus infection. These sequence variations seem to occur as an adaptive response of hepatitis C virus to evade host immunity and may play a major role in the establishment of persistent infection and in the occasional flare-up of hepatitis.

Aged↗
PubMed1992

Clinical backgrounds of the patients having different types of hepatitis C virus genomes.

Hepatitis C virus (HCV) genomes were recently detected in biological materials, and variations of nucleotide sequences were reported. In the present study, typing of the HCV genomes was performed in 91 HCV-RNA-positive patients and the clinical features of patients with different types of HCV were compared. From the nucleotide sequences of the cDNA fragments, HCV can be divided into at least two types: HCV-K1-PT and HCV-K2. All cDNAs amplified from 91 patients were hybridized with cDNA probes of either HCV-K1-PT or HCV-K2. HCV-K1-PT was found in about 80% of the patients, and HCV-K2 was found in about 20% of the patients. These results indicate that types of HCV are limited to two types, i.e., K1-PT and K2, and the major type is HCV-K1-PT, at least in Japan. Detection rate of antibodies to C-100-3 protein were not different between the patients having HCV-K1-PT and HCV-K2, indicating that the antibodies may develop in HCV-related patients without relation to the types of the HCV genomes. Prevalence of the two types of HCV were nearly the same in various forms of NANB-related liver disease. However, the prevalence was somewhat different in alcoholic liver disease. HCV-K2 was found in patients younger than the patients with HCV-K1-PT. Frequency of a history of blood transfusion tended to be lower and the initial response to interferon treatment was clearly better in patients having HCV-K2 versus patients having HCV-K1-PT. These results suggest the possibility that clinical features due to HCV-K1 may be somewhat different from those due to HCV-K1-PT. However, the number of patients examined was too small to allow a definite conclusion, indicating a necessity for further study with a larger number of patients.

DNA, Viral↗
PubMed1991

Hepatitis C virus RNA genome in plasma of patients with non-A, non-B hepatitis.

Recently, the assay system of anti-hepatitis C virus antibody (HCV-Ab) was developed. However, there is no clinically useful method to detect hepatitis C virus (HCV) itself. The authors recently developed a method to detect the HCV-RNA genome in plasma using polymerase chain reaction (PCR). In the present study, the specificity of this assay in detecting HCV infection was investigated. Freshly obtained 1 ml plasma specimens from 100 patients with various liver diseases and from 11 control subjects were studied. In patients with non-A, non-B (NANB) hepatitis-related liver diseases, HCV-RNA was detected in 2 out of 7 cases of acute hepatitis, in 29 out of 31 cases of chronic hepatitis, in 17 out of 21 cases of cirrhosis and in 2 out of 6 cases of hepatocellular carcinoma. On the other hand, no HCV-RNA was detected in 15 cases of various types of alcoholic liver diseases, in 12 cases of hepatitis B related liver diseases, and in 11 controls. HCV-RNA was detected in 2 of 6 drinkers with chronic hepatitis. The prevalence of HCV-RNA was not closely related to a history of blood transfusions. These results suggest that our method for HCV-RNA is specific for HCV infection and HCV infection is the likely etiology of most chronic NANB hepatitis cases. The clinical usefulness of our method is illustrated by the fact that we were able to study 100 patients and needed only 1 ml plasma per HCV-RNA assay.

Electrophoresis, Polyacrylamide Gel↗
PubMed1991

Genotyping of the aldehyde dehydrogenase 2 (ALDH2) gene using the polymerase chain reaction: evidence for single point mutation in the ALDH2 gene of ALDH2-deficiency.

About half of all Japanese lack the activity of aldehyde dehydrogenase 2 (ALDH2), and suffer a flush after alcohol intake due to the marked elevation of blood acetaldehyde concentration. The cause of ALDH2 deficiency is thought to be a single point mutation in codon 487 of the ALDH2 gene. However, this mutant ALDH2 gene has not yet been cloned and sequenced. We amplified and cloned the exon 12 of the ALDH2 gene using polymerase chain reaction (PCR), and revealed that normal GAA coding glutamic acid is replaced for AAA coding lysine in codon 487 of the mutant ALDH2 gene. Based on this finding, we performed the genotyping of the ALDH2 gene using PCR and allele-specific oligonucleotide probes. The genotypes of 13 subjects with ALDH2-active phenotype were all homozygous for the normal ALDH2 gene (ALDH2(1)), while in 9 subjects with ALDH2-deficient phenotype 2 subjects were homozygous for the mutant ALDH2 gene (ALDH2(2)) and the other 7 subjects were heterozygous for both genes, indicating that the mutant ALDH2 gene is dominant. In 20 normal control subjects, the prevalence of ALDH2(1)/ALDH2(1), ALDH2(1)/ALDH2(2) and ALDH2(2)/ALDH2(2) was 45%, 45% and 10% respectively. On the other hand, in 36 alcoholic liver disease patients, the prevalence of the genotypes was 83%, 17% and 0%. These results confirmed the previous observation that the incidence of ALDH2 deficiency is much lower in alcoholic liver disease patients than in the general population, and suggested that most of the ALDH2 deficient patients with alcoholic liver disease are heterozygous for the normal and mutant ALDH2 genes.

Aldehyde Dehydrogenase↗
PubMed1991

Typing of hepatitis C virus genomes by restriction fragment length polymorphism.

Recently, we reported that hepatitis C virus (HCV) can be classified genetically into two types, HCV-K1 and HCV-K2, which show 67% and 71% identity at the nucleotide and amino acid sequence levels in a 340 bp region which encodes the NS5 gene Gly-Asp-Asp motif. To develop a rapid method to classify the genomes of HCV isolates, we identified restriction fragment length polymorphisms (RFLPs) in reverse transcriptase-polymerase chain reaction products encoding a portion of the NS5 gene. AluI and AccII enabled HCV to be classified into the K1 and K2 types, and Sau96I enabled classification into the K1 type, and the K2a and K2b subtypes. These RFLPs also generally allow Japanese isolates to be distinguished from the prototype (PT, an isolate from the U.S.A.), which is a K1 type. Sequence analysis of the 5'-untranslated regions of Japanese isolates revealed near identity between the K1 type and PT, and 93 to 94% identity between the K1 and K2 types, indicating that there are type K1- and K2-specific RFLPs in this region. Our results suggest that the nucleotide sequences of the K1 and K2 types are different throughout the HCV genome. The incidence of HCV types K1, K2a and K2b, and PT in 50 samples was 74%, 16%, 8% and 2%, respectively.

Base Sequence↗
PubMed1991

Acetaldehyde metabolism in different aldehyde dehydrogenase-2 genotypes.

In order to clarify the relationships between acetaldehyde (Ac-CHO) metabolism and low Km (mitochondrial) aldehyde dehydrogenase (ALDH2) genotypes, hepatic ALDH2 activity was determined and serial changes of blood Ac-CHO levels after ethanol administration were analyzed in the individuals homozygous for the normal ALDH2 genes, heterozygous for the normal and mutant ALDH2 genes, and homozygous for the mutant ALDH2 genes. Genomic DNA was extracted from white blood cells and genotyping of ALDH2 was performed using the polymerase chain reaction technique and slot blot hybridization with synthesized oligonucleotide probes specific to the normal and mutant ALDH2 genes. ALDH2 activity was not detectable in the liver in two cases of the mutant homozygote. In four out of eight cases of the heterozygote, hepatic ALDH2 activity was measurable, although the activity was lower compared with that in the normal homozygote. Blood ethanol levels after alcohol administration were not different among the three different ALDH2 genotypes. Blood Ac-CHO levels after drinking of alcohol were significantly higher in the heterozygotes and the mutant homozygotes than in the normal homozygotes. The levels after a moderate amount of ethanol (0.8 g/kg of body weight) in a case of the mutant homozygote were not different from those of the heterozygotes. However, the levels after a small amount of ethanol (0.1 g/kg of body weight) were significantly higher in the mutant homozygotes than in the heterozygotes. These results indicate that hepatic ALDH2 activity is lacking completely, and metabolism of Ac-CHO in the liver is severely impaired in the homozygotes of the mutant ALDH2 genes.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetaldehyde↗