Toshihiko Kaneshige

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

BACKGROUND/AIMSnHepatitis 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.nnnMETHODSnThe 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).nnnRESULTSnSeven 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.nnnCONCLUSIONSnThe complexity of the hypervariable region 1 quasispecies may be a factor for predicting IFN inefficacy in patients with chronic hepatitis C.

Quantitative analysis of the usage of human T cell receptor α and β chain variable regions by reverse dot blot hybridization

Abstract We previously developed an adaptor ligation-mediated PCR method to amplify the T cell receptor (TCR) cDNA pools. In the present study we applied reverse dot blot hybridization to PCR-amplified specimens for quantitative analysis of the usage of TCR α and β chain variable (V) region. 44 VA sequence-specific oligonucleotide probes (SSOPs) and 38 VB SSOPs were synthesized corresponding to unique sequences of VA and VB subfamilies. Peripheral blood lymphocytes of ten healthy donors and five T cell clones established from bone marrow cells were examined for VA and VB usage using this method. The results were consistent with those obtained by a colony hybridization method and those by immunofluorescence staining using monoclonal antibodies to VA and VB. Thus, reverse dot blot hybridization for TCR Vα and Vβ is a new, easy and dependable technique useful for analysis of VA and VB usage by human T cells.

Serologic and nucleotide sequencing analyses of a novel DR52-associated DRB1 allele with the DR ‘NJ25’ specificity, designated DRB1∗1307

A novel DR52-associated DRB1* allele, designated DRB1*1307, was encountered in the course of our HLA-DRB1 genotyping study in a Japanese population by PCR-RFLP. Comparison of the nucleotide sequence of its second exon with those of the other known DRB1 alleles revealed that DRB1*1307 was most similar to DRB1*1101, differing by two amino acid substitutions. From a family study, DRB1*1307 was found to segregate with a haplotype of DRB3*0202-DQA1*0501-DQB1*0301, which was also observed with DRB1*1101 in a Japanese population. DRB1*1307 was recognized in three of 652 healthy Japanese controls (gene frequency: 0.24%) with the same DR-DQ haplotype, indicating that DRB1*1307 arose from DRB1*1101 by a gene conversionlike event(s) and/or point mutations. Further, it was also observed that this allele had a strong linkage disequilibrium with HLA-B70 (p < 0.001). This new DRB1*1307 allele was serologically defined as DR NJ25, and it gave an almost identical serologic pattern to DRB1*1406. On sequence comparison, however, no unique amino acid residues conserved in DRB1*1406 and DRB1*1307 but absent in all the other DRB1 alleles could be found, indicating that two amino acid changes at positions 47 and 58 abolished the reactivity against the DR11 antisera.

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.

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

We analyzed the TcR Vβ gene usage beforeand after vaccination with the hepatitis B vaccine sincechanges in the TcR Vβ gene families would beconsidered to provide preliminary evidence of amechanism to prevent HBV infection. Six healthy adultvolunteers received immunizations. TcR Vβ usage,T-cell proliferation, and HLA class II alleles wereexamined in peripheral blood mononuclear cells (PBMC) both before and after vaccination. Furthermore,TcR Vβ usage in postimmunization PBMC was alsocompared with PBMC cultured with recombinant HBsAg(rHBsAg). The level of in vitro T-cell proliferation in the presence of rHBsAg increasedsignificantly (P < 0.01) in PBMC isolated aftervaccinations. Increases in the different TcR Vβgenes were also observed in each individual followingvaccinations, regardless of the similarity in their HLAalleles. Specific HBV-related antigen-responsive T cellswere induced after HB vaccination, without any commonrestriction for the TcR Vβ gene families. The mechanism that helps prevent HBV infection wasthus found to involve multiclonal alterations in the TcRVβ repertoire.

Molecular analysis of T cell receptor Vβ chain to detect leukemia cell clonality in patients by adaptor ligation-mediated polymerase chain reaction

We have developed a simple and rapid method to analyze the clonality of leukemia cells. After three rounds of amplification by adaptor-ligation polymerase chain reaction (PCR), the cDNA is cut with AluI, HaeIII, RsaI, and Sau3AI, and analyzed by polyacrylamide gel electrophoresis. The size of the restriction fragments is compared to that of the published restriction fragments size each TCR-beta subfamily V region. The sensitivity of adaptor-ligation PCR restriction enzyme analysis (AL-PCR-REA) was 10(-4) MOLT-4 T-ALL cell population in the normal peripheral blood lymphocytes (PBL). Application of AL-PCR-REA to PBL and bone marrow (BM) cells from eight clinical leukemia samples indicated that a detection sensitivity was rather low, but revealed the clonality of all eight clinical samples. This AL-PCR-REA method can detect clonality without the need for either radioisotopes or sequencing procedures.