Publications (25)75.02 Total impact
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Article: Sequential immunological analysis of HBV/HCV co-infected patients during Peg-IFN/RBV therapy.
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ABSTRACT: BACKGROUND: The immunopathogenesis of dual chronic infection with hepatitis B virus and hepatitis C virus (HBV/HCV) remains unclear. The in vivo suppressive effects of each virus on the other have been reported. In this study we aimed to analyze the virological and immunological parameters of HBV/HCV coinfected patients during pegylated interferon/ribavirin (Peg-IFN/RBV) therapy. METHODS: One patient with high HBV-DNA and high HCV-RNA titers (HBV-high/HCV-high) and 5 patients with low HBV-DNA and high HCV-RNA titers (HBV-low/HCV-high) were enrolled. Twenty patients monoinfected with HBV and 10 patients monoinfected with HCV were enrolled as control subjects.. In vitro cultures of Huh 7 cells with HBV/HCV dual infection were used to analyze the direct interaction of HBV/HCV. RESULTS: Direct interaction of HBV clones and HCV could not be detected in the Huh-7 cells. In the HBV-high/HCV-high-patient, the HCV-RNA level gradually declined and HBV-DNA gradually increased during Peg-IFN/RBV therapy. Activated CD4- and CD8-positive T cells were increased at 1 month of Peg-IFN/RBV-therapy, but HBV-specific IFN-γ-secreting cells were not increased and HBV-specific interleukin (IL)-10 secreting cells were increased. The level of HBV- and HCV-specific IFN-γ-secreting cells in the HBV-high/HCV-high-patient was low in comparison to that in the HBV- or HCV-monoinfected patients. In the HBV-low/HCV-high-patient, HCV-RNA and HBV-DNA rapidly declined during Peg-IFN/RBV therapy. Activated CD4- and CD8-positive T cells were increased, and HBV- and HCV-specific IFN-γ-secreting cells were also increased during Peg-IFN/RBV-therapy. CONCLUSION: The immunological responses of the HBV-high/HCV-high patient were low in comparison to the responses in HBV and HCV monoinfected patients. Moreover, the response of immune cells in the HBV-high/HCV-high patient during Peg-IFN/RBV therapy was insufficient to suppress HBV and HCV.Journal of Gastroenterology 05/2012; · 4.16 Impact Factor -
Article: Rapid reduction of hepatitis C virus-Core protein in the peripheral blood improve the immunological response in chronic hepatitis C patients.
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ABSTRACT: Aim: The extracellular hepatitis C virus (HCV)-antigen, including HCV-Core protein, can suppress immune cells. Recently, the efficacy of double filtration plasmapheresis (DFPP) for chronic hepatitis C (CHC) was reported. However, the mechanism of efficacy of DFPP might not be only the reduction of HCV but also the effect of immune cells via direct and/or indirect mechanisms. The aim of this study is to analyze the virological and immunological parameters of difficult-to-treat HCV patients treated with DFPP combined with Peg-interferon and RBV (DFPP/Peg-IFN/RBV) therapy. Methods: Twelve CHC patients were enrolled and treated with DFPP/Peg-IFN/RBV therapy. The immunological, virological and genetic parameters were studied. Results: All patients (4/4) treated with the major IL28B allele (T/T) could achieve complete early virological response (EVR). The amounts of HCV-Core antigen in the peripheral blood of EVR patients treated with DFPP/Peg-IFN/RBV rapidly declined in comparison to those of late virological response (LVR) patients treated with DFPP/Peg-IFN/RBV and EVR patients treated with Peg-IFN and RBV (Peg-IFN/RBV). The amount of IFN-γ produced from peripheral blood gradually increased. On the other hand, the amount of IL10 gradually decreased in the EVR patients. The frequencies of HCV-Core binding on CD3+ T cells rapidly declined in EVR patients treated with DFPP/Peg-IFN/RBV therapy. Moreover, the distributions of activated CD4(+) and CD8(+) T cells and CD16-CD56 high natural killer cells were significantly changed between before and after DFPP. Conclusions: The rapid reduction of HCV-Core antigens and changes in the distribution of lymphoid cells could contribute to the favorable immunological response during DFPP/Peg-IFN/RBV therapy.Hepatology Research 09/2011; 41(12):1153-68. · 2.20 Impact Factor -
Article: Lymphotropic HCV strain can infect human primary naïve CD4+ cells and affect their proliferation and IFN-γ secretion activity.
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ABSTRACT: Lymphotropic hepatitis C virus (HCV) infection of B and T cells might play an important role in the pathogenesis of hepatitis C. Recently, we showed that a lymphotropic HCV (SB strain) could infect established T-cell lines and B-cell lines. However, whether HCV replication interferes with cell proliferation and function in primary T lymphocytes is still unclear. The aim of this study was to analyze whether HCV replication in primary T lymphocytes affected their development, proliferation, and Th1 commitment. SB strain cell culture supernatant (2 × 10(4) copies/ml HCV) was used to infect several kinds of primary lymphocyte subsets. Mock, UV-irradiated SB-HCV, JFH-1 strain, and JFH-1 NS5B mutant, which could not replicate in T cells, were included as negative controls. Carboxyfluorescein succinimidyl ester (CFSE) and CD45RA double staining was used to evaluate the proliferative activity of CD4(+)CD45RA(+)CD45RO(-) naïve CD4(+) cells. Interferon (IFN)-γ and interleukin (IL)-10 secretion assays magnetic cell sorting (MACS) were carried out. Negative strand HCV RNA was detected in CD4(+), CD14(+), and CD19(+) cells. Among CD4(+) cells, CD4(+)CD45RA(+)RO(-) cells (naïve CD4(+) cells) were most susceptible to replication of the SB strain. The levels of CFSE and CD45RA expression gradually declined during cell division in uninfected cells, while HCV-infected naïve CD4(+) cells expressed higher levels of CFSE and CD45RA than Mock or UV-SB infected naïve CD4(+) cells. Moreover, the production of IFN-γ was significantly suppressed in SB-infected naïve CD4(+) cells. Lymphotropic HCV replication suppressed proliferation and development, including that towards Th1 commitment, in human primary naïve CD4(+) cells.Journal of Gastroenterology 02/2011; 46(2):232-41. · 4.16 Impact Factor -
Article: Hepatitis B virus replication could enhance regulatory T cell activity by producing soluble heat shock protein 60 from hepatocytes.
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ABSTRACT: HBcAg-specific regulatory T (T(reg)) cells play an important role in the pathogenesis of chronic hepatitis B. Soluble heat shock proteins, especially soluble heat shock protein 60 (sHSP60), could affect the function of T(reg) cells via Toll-like receptor. We analyzed the relationship between soluble heat shock protein production and hepatitis B virus (HBV) replication with both clinical samples from HBeAg-positive patients with chronic hepatitis B (n= 24) and HBeAb-positive patients with chronic hepatitis B (n= 24) and in vitro HBV-replicating hepatocytes. Thereafter, we examined the biological effects of sHSP60 with isolated T(reg) cells. The serum levels of sHSP60 in patients with chronic hepatitis B were statistically significantly higher than those in patients with chronic hepatitis C (P<.01), and the levels of sHSP60 were correlated with the HBV DNA levels (r = 0.532; P<.001) but not with the alanine aminotransferase levels. Moreover, the levels of sHSP60 in HBV-replicating HepG2 cells were statistically significantly higher than those in control HepG2 cells. Preincubation of CD4(+) CD25(+) cells with recombinant HSP60 (1 ng/mL) statistically significantly increased the frequency of HBcAg-specific interleukin 10-secreting T(reg) cells. The frequency of IL7R(-)CD4(+)CD25(+) cells, the expression of Toll-like receptor 2, and the suppressive function of T(reg) cells had declined during entecavir treatment. The function of HBcAg-specific T(reg) cells was enhanced by sHSP60 produced from HBV-infected hepatocytes. Entecavir treatment suppressed the frequency and function of T(reg) cells; this might contribute to the persistence of HBV infection.The Journal of Infectious Diseases 07/2010; 202(2):202-13. · 6.41 Impact Factor -
Article: Hepatitis C virus infection of T cells inhibits proliferation and enhances fas-mediated apoptosis by down-regulating the expression of CD44 splicing variant 6.
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ABSTRACT: A lymphotropic hepatitis C virus strain (HCV, SB strain, hereafter "SB-HCV") has been shown to infect established T cell lines (Molt-4 and Jurkat) and primary human naive CD4(+) T cells. During T cell development and activation, transient expression of CD44 splicing variant 6 (CD44v6) plays a significant role. SB-HCV was used to infect Molt-4 cells, and their cellular proliferation and CD44 expression was examined. SB-HCV-infected Molt-4 cells expressed a significantly lower level of the CD44v6 isoform. The infected cells could be divided into 2 carboxyfluorescein succinimidyl ester (CFSE) groups, CFSE-high (indicating low proliferation activity; 34.2% of the cells) and CFSE-low (indicating high proliferation activity; 62.5% of the cells), whereas uninfected cells consisted of only a CFSE-low population. Of the CFSE-high cells, 82.4% were positive for the HCV protein NS5A, whereas only 1.2% of the CFSE-low cells were positive for this protein. Among the HCV proteins, NS5A alone caused the down-regulation of CD44v6 expression. After cells were stimulated with phorbol myristate acetate, the amount of phosphorylated mitogen-activated protein (MAP) kinase was significantly reduced in CFSE-high, SB-HCV-infected Molt-4 cells. After Fas ligand stimulation, SB-HCV-infected Molt-4 cells had increased cleavage of caspase 8 and 3 and enhanced apoptosis, compared with the rates of cleavage and apoptosis in control groups, indicating that SB-HCV infection increased Fas-mediated apoptosis. HCV replication in T cells suppresses cellular proliferation and enhances susceptibility to Fas signaling by inhibiting CD44v6 signaling and expression.The Journal of Infectious Diseases 02/2009; 199(5):726-36. · 6.41 Impact Factor -
Article: Analysis of the entire nucleotide sequence of hepatitis B causing consecutive cases of fatal fulminant hepatitis in Miyagi Prefecture Japan.
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ABSTRACT: We encountered five consecutive patients with fulminant hepatitis induced by acute hepatitis B virus (HBV) infection in 2000--2001 in Japan. They had not had previous contact each other, and were referred to us from different hospitals. Although a 69-year-old woman could be rescued by intensive internal treatment, the four patients died. We analyzed the partial (nt 278-646) and entire nucleotide sequences of the HBV obtained from them, and their divergences were 0-0.3% and 0-0.2%, respectively. The results suggested that they had been infected with the same HBV isolates. The isolates belonged to genotype B and subgenotype B2 on the phylogenetic tree analysis (AB302942-AB302946). As for the nucleotides sequences of them, previously reported mutations of G1896A, A1762T, and G1764A were present. Amino acid analysis revealed that previously reported Ile97Leu and Pro130Non-Pro in the core region and Trp28Stop in the precore region were present. As for the entire nucleotide sequences among B2, AB302942 showed low divergences with AF121245 and AB073834 (1.7%), and X97850 from patients with fulminant hepatitis (3.2%). We compared the two consensus nucleotides derived from AB302942 and X97850 (fulminant hepatitis) versus AY121245 and AB073834 (non-fulminant hepatitis), which revealed a difference in nt 1,504 located in the P and X region. Nucleotide 1,504 was C for isolates from fulminant hepatitis and G for non-fulminant hepatitis, and it was recognized among most of the isolates belonging to B2 registered on GenBank. Further studies could disclose the mechanism of severe inflammation of liver that finally leads to fulminant hepatitis.Journal of Medical Virology 07/2008; 80(6):967-73. · 2.82 Impact Factor -
Article: Dynamics of immature subsets of dendritic cells during antiviral therapy in HLA-A24-positive chronic hepatitis C patients.
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ABSTRACT: The cellular immune response is important in chronic hepatitis C (CHC). To better understand its mechanism, we examined dendritic cells (DCs) and hepatitis C virus (HCV)-specific cytotoxic T cells (CTLs), which are thought to contribute to liver injury and viral clearance. CHC patients received 24 weeks of interferon-alpha-based antiviral therapy. We analyzed time-sequential frequencies of peripheral DCs, classified as myeloid DCs (mDCs) or plasmacytoid DCs (pDCs), together with peptide major histocompatibility class I tetramers, epitope specific for HCV core 129-137 (t*24/c129) or HCV NS3 1296-1304 (t*24/ns1294), directly ex vivo. The mDC and pDC populations changed in parallel (P < 0.05), showing a significant transient decrease at weeks 12 and 16 during the therapy, and then recovering. However, neither of the tetramer results showed a direct correlation with the kinetics of peripheral DCs. There is an apparent effect of antiviral therapy or a subsequent reduction of HCV on host immunity, but the effect may not include the induction of CTLs in CHC.Journal of Gastroenterology 08/2006; 41(8):758-64. · 4.16 Impact Factor -
Article: Mechanism of T cell hyporesponsiveness to HBcAg is associated with regulatory T cells in chronic hepatitis B.
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ABSTRACT: To study the mechanisms of hyporesponsiveness of HBV-specific CD4+ T cells by testing TH1 and TH2 commitment and regulatory T cells. Nine patients with chronic hepatitis B were enrolled. Peripheral blood mononuclear cells were stimulated with HBcAg or HBsAg to evaluate their potential to commit to TH1 and TH2 differentiation. HBcAg-specific activity of regulatory T cells was evaluated by staining with antibodies to CD4, CD25, CTLA-4 and interleukin-10. The role of regulatory T cells was further assessed by treatment with anti-interleukin-10 antibody and depletion of CD4+CD25+ cells. Level of mRNAs for T-bet, IL-12R beta2 and IL-4 was significantly lower in the patients than in healthy subjects with HBcAg stimulation. Although populations of CD4+CD25highCTLA-4+ T cells were not different between the patients and healthy subjects, IL-10 secreting cells were found in CD4+ cells and CD4+CD25+ cells in the patients in response to HBcAg, and they were not found in cells which were stimulated with HBsAg. Addition of anti-IL-10 antibody recovered the amount of HBcAg-specific TH1 antibody compared with control antibody (P < 0.01, 0.34% +/- 0.12% vs 0.15% +/- 0.04%). Deletion of CD4+CD25+ T cells increased the amount of HBcAg-specific TH1 antibody when compared with lymphocytes reconstituted using regulatory T cells (P < 0.01, 0.03% +/- 0.02% vs 0.18% +/- 0.05%). The results indicate that the mechanism of T cell hyporesponsiveness to HBcAg includes activation of HBcAg-induced regulatory T cells in contrast to an increase in TH2-committed cells in response to HBsAg.World Journal of Gastroenterology 07/2006; 12(27):4310-7. · 2.47 Impact Factor -
Article: Analysis of the entire nucleotide sequence of hepatitis B virus genotype B in the Philippines reveals a new subgenotype of genotype B.
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ABSTRACT: The entire nucleotide sequences were determined for hepatitis B virus (HBV) genotype B (HBV/B) genomes extracted from five patients in the Philippines and designated GenBank AB219426, AB219427, AB219428, AB219429 and AB219430. The serotype of the first four isolates was ayw and that of GenBank AB219430 was adw. Divergences of entire sequences were 1.0-2.0 % between the first four isolates and 3.8-4.2 % between these four and GenBank AB219430. Phylogenetic-tree analysis revealed that, worldwide, HBV/B comprises five subgenotypes: B1, B2, B3, B4 and the new Philippines group, designated B5. Divergences of the entire genome sequences between four isolates in subgenotype B5 and isolates from other countries (subgenotypes) were 4.4-4.8 % with Vietnam (B4), 2.9-3.5 % with Indonesia (B3), 4.7-5.1 % with China (B2) and 5.4-6.0 % with Japan (B1). Similarly, GenBank AB219430 showed the lowest divergences: 3.4 % with the isolate from Indonesia (B3), 5.0 % with Vietnam (B4), 5.4 % with China (B2) and 6.1 % with Japan (B1). This is the first report of entire nucleotide sequences of HBV/B from the Philippines and the results show that these sequences belong to a new subgenotype, B5. The present study identified that HBV/B isolates throughout the world are divided genetically into five subgenotypes, the relationships between geographical distances and the genetic distances of HBV/B being well-correlated.Journal of General Virology 06/2006; 87(Pt 5):1175-80. · 3.36 Impact Factor -
Article: Th1 response during ribavirin and interferon-alpha combination therapy in chronic hepatitis C.
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ABSTRACT: Ribavirin and interferon-alpha induce Th1 polarization of human CD4+ T cells. The study was conducted to investigate the whether cellular immune response during ribavirin/interferon-alpha therapy is associated with viral eradication by examining mRNA expression of molecules relevant to Th1 and Th2 polarization in CD4+ cells of 13 patients with chronic hepatitis C (seven patients with sustained viral response and six with transient response). Peripheral CD4+ T lymphocytes at 0, 4 and 24 weeks of treatment were tested. There were no significant differences in the mRNA levels at each point of time of the treatment between patients with sustained viral response and those with transient response. The percent increase in mRNA level of the IL-12R beta2 chain from the baseline to the end of the treatment was significantly higher in patients with sustained viral response (15.3+/-6.1%) than in those with transient response (-1.6+/-4.7%, p<0.05). There was no significant difference in percent changes in level of IL-12R beta1 chain mRNA between the two groups. In conclusion, the results of this study indicate that the increase of Th1 response is related to the inflammatory activity in the liver and possibly to ribavirin and interferon-alpha therapy. It is also suggested that the measurement of Th1 response has the potential to distinguish patients with relapse from those with sustained virus response.Hepatology Research 03/2006; 34(2):104-10. · 2.20 Impact Factor -
Article: [Immune dysregulation mediated by hepatitis C virus infection].
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ABSTRACT: Hepatitis C virus, which is non-cytopathic, establishes persistent infection in majority of patients after acute infection, causing various degrees of clinical liver disease. To escape and survive, hepatitis C virus may take ingenious strategies. Hepatitis C virus gene products interact host proteins to evade host immune responses in addition to the appearance of quasispecies. Against hepatitis C virus infection, host may avoid extensive tissue damage by inducing the activity of regulatory T cells. Insights into this mechanism of immune regulation may help to future development of novel therapies against hepatitis C virus.Nihon Hansenbyo Gakkai zasshi = Japanese journal of leprosy: official organ of the Japanese Leprosy Association 10/2005; 74(3):181-4. -
Article: Recovery of functional cytotoxic T lymphocytes during lamivudine therapy by acquiring multi-specificity.
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ABSTRACT: To characterize cytotoxic T lymphocytes (CTLs) that appeared in circulation during lamivudine therapy, we analyzed HBV-specific CTLs using HLA-A24 tetramer and HBcAg-specific Th1 cells in patients receiving lamivudine therapy. Six patients (HLA-A24(+)) with chronic hepatitis B, six patients (HLA-A24(-)) with chronic hepatitis B, and six patients (HLA-A24(+)) with chronic hepatitis C were studied. In addition to known CTL epitopes (C117 and P756), three epitopes were confirmed as CTL epitopes (C23, S89, S226) by chromium release assay and by staining intracellular perforin. CTLs specific for P756 were most frequently found at pre-treatment. During lamivudine therapy, increase in the frequencies of HLA-tetramer(+) cells was found for C117, S89, and S226. Recovery of CTLs was observed earlier in patients with HBeAg(-)/anti-HBe(+) compared with those with HBeAg(+)/anti-HBe(-). HBcAg-specific Th1 cells did not increase significantly up to 8 weeks. T cell lines from patients with chronic hepatitis B had a lower level of proliferation (0- to 24.9-fold expansion by in vitro stimulation) and a higher ability to produce interferon-gamma (0-84% except for S89), while perforin-positive cells showed low frequencies (0-50% except for S89). In conclusion, these results suggest that lamivudine therapy induces mainly CTLs that were less frequent before the therapy. Since recovered CTLs maintained the ability to produce interferon-gamma in response to peptides, these CTLs apparently contribute to the efficacy of lamivudine therapy in patients with hepatitis B.Journal of Medical Virology 12/2004; 74(3):425-33. · 2.82 Impact Factor -
Article: Ribavirin upregulates interleukin-12 receptor and induces T cell differentiation towards type 1 in chronic hepatitis C.
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ABSTRACT: The mechanisms of ribavirin as an immune modulator have not been fully revealed, contrary to its clinical benefit in chronic hepatitis C. Recently, host immune defense, especially cytotoxic T lymphocytes and T helper cells, have been considered to be closely related to the pathophysiology of chronic viral hepatitis. The aim of the present study was to evaluate the function of ribavirin in cellular immunity. Peripheral blood mononuclear cells and total RNA were prepared from chronic hepatitis C patients. To evaluate the polarization of T helper cells, we performed intracellular cytokine assay and quantified the production of key cytokines. mRNA levels of interleukin-12 receptor (IL-12R), interferon (IFN)-gamma and interleukin-4 (IL-4) were measured by real time reverse transcription-polymerase chain reaction (RT-PCR). The population of T helper 1 cells increased significantly both in mitogen and hepatitis C virus (HCV) core protein stimulated cells with 0.1 microg/mL of ribavirin. However, the population of T helper 2 cells did not seem to be affected by ribavirin. The level of IL-12R beta2 chain mRNA was also upregulated to 130% after 24 h incubation with 0.1 microg/mL ribavirin (P = 0.037), whereas the beta1 chain did not change significantly under these conditions. In addition, 0.1 microg/mL ribavirin could upregulate the levels of IFN-gamma or IL-4 mRNA in some cases. Ribavirin, perhaps by acting directly on CD4+ T cells, induces T cell differentiation towards type 1, depending on the upregulated signal of the IL-12/IL-12R pathway.Journal of Gastroenterology and Hepatology 06/2004; 19(5):558-64. · 2.87 Impact Factor -
Article: Vectorial transport of bile acids in immortalized mouse bile duct cells.
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ABSTRACT: In ileal epithelial cells, apical sodium-dependent bile acid transporter (ASBT) is responsible for the uptake of bile acids from the lumen. Furthermore, ASBT is expressed in the apical plasma membrane of intrahepatic bile duct cells (BECs). Using cultured immortalized mouse intrahepatic BECs that form monolayers or cysts, vectorial transport of bile acids was studied. [3H]-taurocholic acid ([3H]-TCA) was transported through monolayers transcellularly almost exclusively from the apical to the basolateral side in a Na(+)- and a temperature-dependent manner. Transport of [3H]-TCA was inhibited by 59.3+/-18.6% in the presence of taurochenodeoxycholic acid. Uptake of lysyl fluorescein-conjugated bile acid, Cholyl-[Nepsilon-NBD]-lysine, was seen in a Na(+)- and a temperature-dependent manner from the apical side of BECs that form monolayer or cysts. Reverse transcription-polymerase chain reaction for mRNAs in the cells showed presence of mRNAs for ASBT and farnesoid X receptor (FXR), a nuclear bile acid receptor. In conclusion, intrahepatic BECs transport bile acids mainly from the apical to the basolateral side in concert with ASBT and maybe FXR in the cells.Hepatology Research 11/2003; 27(2):151-157. · 2.20 Impact Factor -
Article: Distribution of the HLA class I allele in chronic hepatitis C and its association with serum ALT level in chronic hepatitis C.
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ABSTRACT: An essential process for resolution of viral infections is the efficient recognition and elimination of intracellular virus. Recognition of viral antigens in the form of short peptides associated with HLA class I molecule is a major task of CD8+ cytotoxic T lymphocytes. In this study, we have evaluated the frequency of the HLA class I alleles in patients with chronic hepatitis C. HLA-B51, -B52, -B55, -B56, -B61, B70, -Cw1, -Cw3, and -Cw4 are less frequent in patients with chronic hepatitis C than in Japanese individuals. The frequency of HLA-A2 is slightly lower in the patients but tends to be higher in patients with normal alanine aminotransferase (ALT) level than in those with elevated ALT level (p = 0.07). Other HLA alleles are not significantly different between two groups. Comparison of HLA homozygosity at HLA-A and -B or -C or at two or three loci did not show a significant association with levels of serum ALT or with the clinical outcome of interferon therapy in patients with hepatitis C. These results suggest a possibility that the alterations of host response, which depends on genetic background, influence disease activities of HCV infection.The Tohoku Journal of Experimental Medicine 11/2003; 201(2):109-17. · 1.24 Impact Factor -
Article: Idiotype-anti-idiotype-based noncompetitive enzyme-linked immunosorbent assay of ursodeoxycholic acid 7-N-acetylglucosaminides in human urine with subfemtomole range sensitivity.
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ABSTRACT: We have established a noncompetitive enzyme-linked immunosorbent assay (ELISA) for the group-specific determination of 7-N-acetylglucosaminide of ursodeoxycholic acid (UDCA 7-NAG) and its glycine- and taurine-amidated metabolites (UDCA 7-NAGs) in human urine. These metabolites are expected to be a diagnostic marker for patients with primary biliary cirrhosis (PBC). This assay is based on the idiotype-antiidiotype reaction where the analyte was captured by an excess amount of anti-UDCA 7-NAG antibody, and the unoccupied paratope was blocked with a beta-type antiidiotype antibody. The hapten-occupied antibody was then selectively detected with a biotin-labeled alpha-type antiidiotype antibody. The amount of bound biotin, increasing proportionally to the increase in the analyte, was colorimetrically determined using a peroxidase-labeled streptavidin. This assay provided subfemtomole range sensitivity (detection limit 118 amol) and allowed group-specific measurement of the UDCA 7-NAGs in urine without any pretreatment. The present ELISA revealed that significant amounts of UDCA 7-NAGs are excreted even in healthy subjects. Daily excretion rates for healthy males were determined to be 246+/-184 (S.D.) microg (n=5) as the glycine-amidated UDCA 7-NAG equivalent. Randomly collected urine specimens from patients with PBC (n=7) were also measured, and the assay values (standardized to creatinine excretion) ranged from 1.82 to 13.4 microg/mg Ucre with the average of 5.41+/-4.53 (S.D.) microg/mg Ucre.Journal of Immunological Methods 02/2003; 272(1-2):1-10. · 2.20 Impact Factor -
Article: A monoclonal antibody-based enzyme-linked immunosorbent assay of glycolithocholic acid sulfate in human urine for liver function test.
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ABSTRACT: Urinary levels of sulfated metabolites of lithocholic acid (LCA) are expected to be a useful index of liver function. Thus, a sensitive, specific, and feasible enzyme-linked immunosorbent assay (ELISA) of these sulfated LCA metabolites (LCA-Suls) should be established. A newly generated monoclonal antibody specific to glycolithocholic acid sulfate (glycine-amidated LCA-Sul (GLCA-Sul)) was immobilized on microtiter plates via a second antibody. A urine specimen and an alkaline phosphatase-labeled antigen were added to the plate, which was then incubated at room temperature for 3h. After this competitive reaction, bound enzyme activity was measured colorimetrically using p-nitrophenyl phosphate as a substrate. The detection limit for GLCA-Sul was 0.4 pg/assay. Nonamidated LCA-Sul and taurine-conjugated LCA-Sul showed 40 and 11% cross-reactivities, respectively, while 3-sulfates of cholic acid (CA; 0.02%), chenodeoxycholic acid (CDCA; 0.63%), and deoxycholic acid (DCA; 2.2%) exhibited very low cross-reactivities. Applicability of the ELISA system to clinical samples was well validated by parallelism, recovery test, and intra/inter-assay variance. Enzymatic deconjugation with bile acids sulfatase resulted in dramatically decreased urinary levels, supporting the specificity of the ELISA toward GLCA-Sul. The mean GLCA-Sul levels in early morning urine from healthy volunteers were 314 ng/mg Ucre (males: n=16) and 507 ng/mg Ucre (females: n=9). Patients with liver diseases, including chronic hepatitis (CH) and liver cirrhosis (LC) exhibited significantly higher values (mean 5222 ng/mg Ucre: n=21). The present 'monoclonal ELISA' is predicted to be useful as a novel noninvasive diagnostic tool for liver function and hepatobiliary diseases.Steroids 10/2002; 67(10):827-33. · 2.83 Impact Factor -
Article: Analysis of the entire nucleotide sequence of hepatitis B virus: characteristics of HBeAg-positive asymptomatic carriers, HBeAb positive asymptomatic carriers and patients with liver cirrhosis.
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ABSTRACT: Entire nucleotide sequences of the HBV genome typical for various stages of HBV carriers are currently unknown. Comparison between conserved sequences in HBeAg-positive asymptomatic carriers (HBeAg ASCs) and mutations characteristic for HBeAb-positive asymptomatic carriers (HBeAb ASCs) are of clinical importance. In this study, we determined the entire nucleotide sequences of the HBV genome of patients infected with genotype C (HBeAg ASCs, 11 cases; HBeAb ASCs, seven cases; patients with liver cirrhosis (LC), five cases). Mutations in the entire nucleotide sequences were found more frequently in HBeAb ASCs than in HBeAg ASCs. In the precore/core (preC/C) region, amino acid mutations were more frequent in HBeAb ASCs (3.03%) than in HBeAg ASCs (0.00%) and patients with LC (0.69%). It was suggested that the mutations in the preC/C region had a close relationship with clinical status of HBV carriers. Mutations of leucine to isoleucine at a.a. 100 of the core region and of threonine to serine at a.a. 340 of the polymerase region were found frequently in HBeAb ASCs. In patients with LC, it was suggested that defective interfering particles (DI particles) play a role in the progression of stages. We conclude that attention should be given to mutations at a.a. 340 of the polymerase protein in addition to core protein.Hepatology Research 09/2002; 23(4):251. · 2.20 Impact Factor -
Article: Up-regulation of CD11a (LFA-1) expression on peripheral CD4+ T cells in primary biliary cirrhosis.
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ABSTRACT: Up-regulation of CD11a expression on CD4+ T lymphocytes is considered to be one of the mechanisms involved in the initiation of the Th-1-mediated immune response. In this study, peripheral blood mononuclear cells from patients with primary biliary cirrhosis (PBC) were evaluated for CD11a(high) CD2(low) T cells and populations of type 1 (Th-1) and type 2 (Th-2) helper T cells. CD11a(high) CD2(low) T cells were found in PBC (7/15) and in active rheumatoid arthritis (4/4), but not in chronic hepatitis C (0/5) or in healthy subjects (0/6). The population of Th-1 had a positive correlation with that of CD4+ CD11a(high) CD2+ cells in patients with PBC (P = 0.034). The serum levels of interferon-gamma also had a weak correlation with the population of CD4+ CD11a(high) CD2(low) cells (P = 0.050). There was no statistically significant correlation of Th-2 population (P = 0.295) or serum interleukin-4 level (P = 0.685) with the population of CD4+ CD11a(high) CD2(low) cells. These results suggest that CD4+ CD11a(high) cells play a role in Th-1-predominance and in the autoimmune process of PBC.Digestive Diseases and Sciences 07/2002; 47(6):1209-15. · 2.12 Impact Factor -
Article: Gene expression of interleukin-12 receptor beta2 chain and Th1 population of peripheral blood mononuclear cells in chronic hepatitis C.
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ABSTRACT: Gene expression of interleukin 12-receptor beta2 chain mRNA in peripheral blood mononuclear cells (PBMCs) was examined in patients with chronic hepatitis C (n=7) and in healthy control subjects (n=6) by semi-quantitative RT-PCR. The level of interleukin 12-receptor beta2 chain mRNA was higher in patients with chronic hepatitis C than in healthy subjects (P=0.032). The level of interleukin 12-receptor beta2 chain mRNA had a weak correlation with the ratio of Th1 to Th2 populations (r=0.714, P=0.020). There was a tendency for the level of interleukin 12-receptor beta2 mRNA to increase both in chronic hepatitis C (P=0.109) and in healthy volunteers (P=0.144) after the incubation of PBMCs with interferon-alpha in vitro. During interferon-alpha administration to the patients with chronic hepatitis C, the level of interleukin 12-receptor beta2 chain mRNA in PBMCs was increased in all four cases. Although this is a preliminary study with a small sample size, our results suggest that the level of interleukin 12-receptor beta2 chain mRNA is higher than normal in patients with chronic hepatitis C and can be further enhanced by interferon therapy.Hepatology Research 05/2002; 22(4):270-277. · 2.20 Impact Factor
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1991–2010
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Tohoku University
- • Department of Gastroenterology
- • Graduate School of Medicine
- • Division of Internal Medicine
Sendai-shi, Miyagi-ken, Japan
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