Circulating cell-free DNA as a predictive marker for distant metastasis of hepatitis C virus-related hepatocellular carcinoma.

In a previous study, we showed that levels of cell-free DNA (cfDNA) were significantly higher in sera of patients with hepatocellular carcinoma (HCC) associated with hepatitis C virus (HCV) than in sera of non-HCC patients with HCV. To confirm this finding, we analysed serum cfDNA levels in a cohort of 96 patients with HCV-related HCC and in 100 HCV carriers without known HCC. Again we found that serum cfDNA levels were significantly higher in HCC patients than in HCV carriers (115.9+/-98.3 vs 34.4+/-40.4 ng ml(-1) (mean+/-s.d.), P<0.0001). Of 87 eligible patients who underwent curative hepatectomy, those with a high cfDNA level had a significantly shorter overall survival (OS) time than those in whom the cfDNA level was not high. Cox proportional hazards model showed the cfDNA level to be an independent prognostic factor for OS and cancer recurrence in distant organs. Our results suggest that the serum cfDNA level reflects the metastatic potential of HCV-related HCC and that it can be a useful predictive biomarker for distant metastasis after curative surgery.

Rapid and simultaneous HLA class I (-A, -B and -C loci) DNA typing using the microtitre plate-reverse hybridization assay (MRHA).

We have established a precise, rapid, simple and practical HLA class I DNA typing method using the microtitre plate-reverse hybridization assay (MRHA), which enables us to perform simultaneous DNA typing of the HLA-A, -B and -C loci using the same PCR parameters and hybridization conditions. PCR-amplified products for the HLA-A, -B and -C loci were hybridized, respectively, with sequence-specific oligonucleotide (SSO) probes, which were immobilized covalently onto a microtitre plate, in hybridization buffer containing formamide at 37 degrees C. After washing at room temperature, the bound PCR products were detected by peroxidase-conjugate streptavidine followed by colour development such as enzyme immunoassay (EIA). In addition to the simple thermoregulation for hybridization and postwashing, strong positive signals, low background and high reproducibility, this DNA typing method enabled simultaneous typing of the HLA-A, -B and -C loci using a single microtitre plate as in HLA serotyping. The assignment of the HLA genotype was easily achieved by automated colorimetric reading and computer software, based on the cut-off value (threshold) established for each probe. For routine HLA class I typing, it may be possible to replace serological typing with the HLA class I DNA typing system using our MRHA method.

Rapid HLA class I DNA typing using microtiter plate-reverse hybridization assay (MRHA) by simple thermoregulation: high-resolution subtyping of the HLA-A2 and -B40 antigen groups.

We have established a precise, rapid, simple and economical subtyping method for alleles encoding the HLA-A2 and -B40 antigens using microtiter plate-reverse hybridization assay (MRHA), which is based on the general principle of HLA oligotyping by reverse dot blot hybridization. Amino-modified sequence-specific oligonucleotide (SSO) probes were immobilized covalently onto a carboxylate-modified microtiter plate. In order to perform high-resolution subtyping of the HLA-A2 and -B40 antigen groups, the alpha1 and alpha2 domain regions were amplified using a pair of group-specific primers composed of an unlabeled sense primer and a biotinylated antisense primer. PCR-amplified products were hybridized with SSO probes in hybridization buffer containing formamide for 1 hour at 37 degrees C. After washing with 2 X SSC at room temperature, the bound PCR products were detected by alkaline phosphatase-conjugated streptavidine followed by color development. All of 8 HLA-B40 suballeles, all of 2 HLA-B47 suballeles (B40 group-specific primers used in this study allowed also B47 amplification) and 17 out of 21 HLA-A2 suballeles were discriminated. The remaining four HLA-A2 suballeles were determined by analysis after exon 4 amplification. HLA-DNA typing by this method was easily and exactly performed regardless of sample number. The greatest advantages of this technique are strong positive signals obtained, reproducibility and the ease of thermoregulation for hybridization and washing as compared to previously reported microtiter plate hybridization methods.

Involvement of transporter associated with antigen processing 2 (TAP2) gene polymorphisms in hepatitis C virus infection.

BACKGROUND & AIMS: Transporter associated with antigen processing (TAP) has essential roles in the antigen-presenting systems, translocating antigenic peptides from the cytosol into the endoplasmic reticulum. The aim of this study was to clarify whether TAP polymorphisms are involved in hepatitis C virus (HCV) infection. METHODS: The 145 HCV-infected Japanese patients examined in this study were categorized into two groups: 36 carriers with persistently normal alanine transaminase (ALT) values and 109 patients with chronic liver disease (CLD). TAP2 gene phenotypes were determined by means of polymerase chain reaction-restriction fragment length polymorphism, and their frequencies were compared between the two groups. RESULTS: Frequencies of TAP2*0101, *0102, and *0201 were not different between the two groups. However, TAP2*0103 frequency in carriers with normal ALT levels was significantly higher than that in patients with CLD (44% vs. 16%; P = 0.00064, Pc < 0.005). Although the TAP2*0103 allele was tightly linked with class II DRB1*1302-DQB1*0604 haplotype in this study, the TAP2*0103 frequency in the normal ALT group was also significantly higher than that in the CLD group even in DRB1*1302-DQB1*0604-negative patients (31% vs. 10%; P = 0.0076, Pc < 0.05). CONCLUSIONS: These findings suggest that TAP2*0103 may be closely associated with low serum ALT activity in HCV-infected Japanese patients.

Analysis of human T-cell antigen receptor variable beta gene usage following vaccination with recombinant HBsAg.

We analyzed the TcR Vbeta gene usage before and after vaccination with the hepatitis B vaccine since changes in the TcR Vbeta gene families would be considered to provide preliminary evidence of a mechanism to prevent HBV infection. Six healthy adult volunteers received immunizations. TcR Vbeta usage, T-cell proliferation, and HLA class II alleles were examined in peripheral blood mononuclear cells (PBMC) both before and after vaccination. Furthermore, TcR Vbeta usage in postimmunization PBMC was also compared with PBMC cultured with recombinant HBsAg (rHBsAg). The level of in vitro T-cell proliferation in the presence of rHBsAg increased significantly (P < 0.01) in PBMC isolated after vaccinations. Increases in the different TcR Vbeta genes were also observed in each individual following vaccinations, regardless of the similarity in their HLA alleles. Specific HBV-related antigen-responsive T cells were induced after HB vaccination, without any common restriction for the TcR Vbeta gene families. The mechanism that helps prevent HBV infection was thus found to involve multiclonal alterations in the TcR Vbeta repertoire.

Influence of HLA haplotypes on the clinical courses of individuals infected with hepatitis C virus.

The human leukocyte antigen is a crucial genetic factor that initiates or regulates immune response by presenting foreign or self antigens to T lymphocytes. The aim of this study was to investigate whether HLA polymorphism is associated with the onset or progression of liver injury in chronic hepatitis C virus (HCV) infection. We determined HLA class I antigens and class II alleles in 130 hepatitis C virus (HCV)-infected patients (33 carriers with persistently normal alanine transaminase [ALT] values and 97 patients with chronic liver disease [CLD]). HLA class I (A, B) was typed serologically, and class II (DRB1, DQB1) was typed by means of polymerase chain reaction-restriction fragment length polymorphism methods. The frequencies of DRB1*0405 and DQB1*0401 were higher in HCV-infected patients than in uninfected subjects. Among HCV-infected patients, the frequencies of B54, DRB1*0405, and DQB1*0401 were significantly higher in patients with CLD than in those carriers with persistently normal ALT values, whereas DRB1*1302, DRB1*1101, and DQB1*0604 were more frequently found in carriers with persistently normal ALT values than in patients with CLD. From extended haplotype analyses, in carriers with B54-DRB1*0405-DQB1*0401 haplotype, the risk of having liver injury was 13.2 times greater than in carriers with DRB1*0405-DQB1*0401 but without B54 [P = 0.0015, Haldane odds ratio = 13.2 (95% confidence interval, 1.7-103.8)]. In contrast, carriers with B44-DRB1*1302-DQB1*0604 had a 12.7-fold lower relative risk of developing liver injury compared to those with the haplotype containing B44 but not DRB1*1302-DQB1*0604 [P = 0.0076, Haldane odds ratio = 0.079 (0.009-0.695)]. Our findings show that extended haplotypes including class I B54 are closely associated with the progression of liver injury, whereas extended haplotypes including class II DRB1*1302-DQB1*0604 are associated with low hepatitis activity in chronic HCV infection.

Complete HLA-A DNA typing using the PCR-RFLP method combined with allele group- and sequence-specific amplification.

We have established a practical method of complete high-resolution typing for all HLA-A alleles using the polymerase chain reaction (PCR)-restriction fragment-length polymorphism (RFLP) technique combined with allele group- and sequence-specific amplification. The second and third exons of the HLA-A gene, in which most allelic variations are observed, were separately amplified by PCRs with 3 and 4 group-specific primer pairs, respectively. Each PCR-amplified product was digested by allele-specific restriction endonucleases and then subjected to electrophoresis on a 10% polyacrylamide gel. In this way, 62 out of 79 HLA-A alleles could be discriminated by the RFLP patterns derived from the genetic polymorphism in the exon 2 and 3 domains. The remaining 17 alleles could be defined unequivocally by either PCR-RFLP analysis after exon 4 amplification or PCR analysis with sequence-specific primers (SSP). By this method, complete HELA-A genotyping for all homozygous and heterozygous combinations can be accomplished, establishing technically simple, economical and practical routine typing of the HLA-A gene, especially for small samples.

Relation of disease activity during chronic hepatitis C infection to complexity of hypervariable region 1 quasispecies.

We studied the heterogeneity in the E2/NS1 hypervariable region 1 of the hepatitis C virus (HCV) genome in relation to the natural course after infection. The subjects were composed of 38 chronic hepatitis C carriers who had been followed for 9 to 218 months after the onset of non-A, non-B (type C) hepatitis, being tested monthly for serum alanine aminotransferase levels. The complexity of the sequence heterogeneity was assessed by single-strand conformation polymorphism analysis. The quasispecies complexity had no relation to the route of infection, the time from infection and the duration of aminotransferase elevation after the onset. However, it had a significant relationship with the degree of aminotransferase elevation in the course of the disease. The quasispecies complexity was directly correlated with the first peak of serum aminotransferase at the onset (r = .48, P < .01) and the mean aminotransferase levels during the period of persistent aminotransferase elevation (r = .58, P < .01). Twenty-three of the 38 patients were further followed for 24 months with biweekly alanine transaminase (ALT) tests. Their aminotransferase levels remained within the normal range during follow-up, and no significant change was seen in the quasispecies complexity after this asymptomatic period. However among the 23 patients, the quasispecies complexity increased in six cases (26%) and decreased in five (22%). A significant direct relation was seen between changes in the quasispecies complexity and the mean aminotransferase levels during the asymptomatic period (r = .55, P = .01). These findings suggest that the development of the HCV quasispecies nature may be related to the severity of the hepatitis in the course of infection.

Haplotypic diversity of DQA1*03 and *05 subtypes differing at amino acid residue 160 encoded in the third exon in 2215 Japanese individuals.

We analyzed the frequencies and haplotypes of DQA1*03 and *05 subtypes, DQA1*03011 or DQA1*0302 and DQA1*0501 or DQA1*0503, respectively, differing only at codon 160 in the non-polymorphic third exon of the DQA1 gene. Of these, 1,862 and 337 individuals selected as DQA1*03- and DQA1*05-positive samples, respectively among 2,215 unrelated Japanese were typed for their nucleotide variation at residue 160 using PCR-SSP. As observed in other populations, all the samples carrying DQA1*03011 (Gene Frequency, GF: 7.8%) were found to share DQB1*0302, whereas those carrying DQA1*0302 (GF: 44.3%) were associated with a variety of DQB1 alleles including DQB1*0302. Both of the DQA1-DQB1 haplotypes with DQA1*03011 and DQA1*0302 carrying DRB1*0406, DQA1*03011-DQB1*0302 and DQA1*0302-DQB1*0302, showed a strong linkage disequilibrium with B62 (p < 0.001, p < 0.05). These results suggested that DQA1*03011 was generated from a single amino acid change at residue 160 in the DQA1*0302-DQB1*0302 haplotype. However, none of the haplotypes with two different DQA1*03 subtypes carrying DRB1*0403,*0405,*0802 and *0901 showed a linkage disequilibrium with any common B-locus antigens, revealing extensive haplotypic diversity of the DQA1*03 group. For example, DRB1*0802 haplotypes showed linkage disequilibria with two different B-locus antigens, B35 and B61 depending on the presence of DQA1*03011 and DQA1*0302, respectively. The GFs of DQA1*0501 and *0503 were 5.1% and 2.7%, respectively. The DQA1*05 associated haplotypes in the DR52-antigen group with DQB1*0301 were divided into two groups, depending on the bimorphism at residue 160. Such a high degree of haplotypic diversity in association with DRB1 and B alleles observed in the DQA1*03 and *05 groups related to amino acid variation at residue 160, which may affect biological function such as the interaction between CD4 and HLA-DQ molecules, seems to reflect selective pressure in the evolutionary process of HLA antigens.

Hepatitis C viral quasispecies in hepatitis C virus carriers with normal liver enzymes and patients with type C chronic liver disease.

Hepatitis C virus (HCV) has been reported to conform to a quasispecies nature, which is most evident in hypervariable regions of the putative envelope 2 domain. The aim of this study was to determine the relationship between the nucleotide complexity and diversity of hypervariable region 1 and various stages of the carrier states. The subjects studied were 20 HCV carriers with normal alanine aminotransferase (ALT) levels, 50 patients with chronic hepatitis who showed elevated ALT levels, 22 with cirrhosis, and 24 with hepatocellular carcinoma. The quasispecies complexity was analyzed by means of polymerase chain reaction-mediated single strand conformation polymorphism (PCR-SSCP). The value of nucleotide diversity was calculated by PCR cloning and sequencing. The number of SSCP bands ranged from 1 to 7, with no significant differences in the mean numbers among the stages of HCV infection. There was no correlation between the amounts of serum HCV RNA and the numbers of SSCP bands. No significant difference was found in the values of nucleotide diversity between carriers with normal ALT levels (mean, 6.6 x 10(-2) per site) and patients with chronic hepatitis (7.7 x 10(-2). These findings suggest that the quasispecies complexity of hypervariable region 1 is independent of the stage of chronic HCV infection.

Hepatitis C viral complexity detected by single-strand conformation polymorphism and response to interferon therapy.

BACKGROUND/AIMS: Hepatitis C virus (HCV) genome heterogeneity by sequence analysis in association with interferon (IFN) inefficacy has been reported. This study was performed to establish a convenient method for detecting the HCV quasispecies complexity and to determine the correlation between the complexity and the responsiveness to IFN therapy in patients with chronic hepatitis C. METHODS: The quasispecies complexity of HCV hypervariable region 1 in patients treated with IFN-alpha was analyzed by polymerase chain reaction-mediated single-strand conformation polymorphism (SSCP). RESULTS: Seven of 25 patients (28%) with low complexity (SSCP band number of < or = 2) were HCV RNA negative after treatment, whereas in 24 patients with high complexity (SSCP band number of > or = 3), the response to IFN was almost insignificant because only 1 patient (4.5%) remained HCV RNA negative after treatment (P < 0.05). Among type 1b patients, IFN therapy was only effective for patients with low amounts of HCV RNA (< or = 10(7.5) copies/mL serum) and low complexity. In contrast, most type 2a patients tended to respond to the therapy with exceptions being those with high amounts of HCV RNA and high complexity. CONCLUSIONS: The complexity of the hypervariable region 1 quasispecies may be a factor for predicting IFN inefficacy in patients with chronic hepatitis C.

NPK15, a tobacco protein-serine/threonine kinase with a single hydrophobic region near the amino-terminus.

A cDNA clone (cNPK15) was isolated from tobacco cells in suspension culture, which encodes a predicted protein kinase of 422 amino acids. The predicted NPK15 protein consists of a hydrophobic region near the amino-terminus, a linker domain and the catalytic domain of a protein-serine/threonine kinase in the carboxyl-half. NPK15 was not found to be closely related to any reported protein, but its putative catalytic domain shares some structural similarity with those of receptor-like protein kinases of plants, such as ZmPK1 from Zea mays and TMK1 from Arabidopsis, even though no receptor-like domain is found in NPK15. Recombinant NPK15 expressed in Escherichia coli as a fusion protein was found capable of autophosphorylation and of phosphorylation of the histone H1 protein on both serine and threonine residues. Upon overexpression of cNPK15 under control of the promoter of cauliflower mosaic virus 35S RNA in tobacco cells, into which it had been introduced by Agrobacterium-mediated transformation, the NPK15 gene acted as a "suicide" gene and blocked proliferation of the host cells. By contrast, such a suicide effect was not observed with the gene for a kinase-negative mutant protein in which the nucleotide sequence for the ATP-binding site had been mutated or with a mutant derivative encoding a protein in which the hydrophobic region had been deleted. Thus, the protein kinase activity of NPK15 and the hydrophobic region of the protein are responsible for the suicide effect. The NPK15 protein kinase seems to be associated with specific cellular functions. Southern blot analysis with cNPK15 as the probe detected several fragments in restriction digests of genomic DNAs from both tobacco and other members of the Solanaceae. This results suggests that NPK15-related genes constitute a small gene family in the genomes of Solanaceae.

Accuracy of the results measured by in vivo 1H-MRS using model solutions.

In in vivo 1H-MRS of human brain, main three metabolites such as N-acetyl-L-aspartate (NAA), creatine/phosphocreatine (PCr/Cr), and choline (Cho) have been observed. In this paper an accuracy of the results measured by in vivo 1H-MRS using above model solutions was discussed. 1.0 mmol/l choline and 1.5 mmol/l creatine/phosphocreatine saline solutions were measured with STEAM method (echo time 270 msec). The peak area of Cho was larger than that of PCr/Cr against their concentrations and was linearly increased with its concentration in the range of 5.8 and 29 mumol/27 cm3. Although both peak areas of Cho and Cr/PCr were varied with Volume-of-interest (VOI) coordinates, the ratio of Cho to PCr/Cr was approximately 1.6.