Clinical implications of measurement of serum nuclear matrix protein levels in patients with liver disease.

BACKGROUND/AIMS: Serum aminotransferase, a sensitive marker of hepatocellular damage, often poorly correlates with the severity of damage. Serum nuclear matrix protein (NMP), a structural protein released from dead cell nuclei, is investigated as a candidate marker of organ damage in liver disease. METHODOLOGY: Serum NMP and aminotransferase levels of 134 patients with various liver diseases and 26 healthy individuals were examined. RESULTS: Patients with chronic viral hepatitis showed slightly higher NMP levels (17.8 U/mL; 95% CI 15.0-20.5 U/mL) than those of healthy individuals (6.05 U/mL; 95% CI 4.82-7.27 U/mL). Their NMP values had no correlation with aminotransferase levels. NMP levels were similar irrespective of liver disease progression, whereas aminotransferase values decreased in parallel with progression. Patients with autoimmune hepatitis or primary biliary cirrhosis who were under an appropriate treatment as well as individuals with fatty liver showed no elevation of serum NMP levels. Patients with acute viral hepatitis showed very high NMP levels (38.8 U/mL; 95%CI 27.6-50.0 U/mL) that correlated with serum aminotransferase levels in their sera. CONCLUSIONS: In chronic liver diseases, the serum NMP level elevates to various degrees independent from the degree of aminotransferase elevation. Serum NMP, putatively representing the number of dead cells, is a candidate as an indicator of organ damage severity in liver disease.

Peripheral lymphocyte subsets vary with stage of hepatitis C virus-associated liver disease.

BACKGROUND/AIMS: Hepatitis C virus induces various clinical features in a host depending on duration of the viral infection. METHODOLOGY: We investigated peripheral lymphocyte subsets in patients with three different stages of hepatitis C virus infection: 5 patients with acute hepatitis, 10 with chronic hepatitis unassociated with cirrhosis, and 10 with cirrhosis. Peripheral lymphocytes were double-stained with multiple fluorescent antibody combinations: anti-CD3 plus anti-gammasigmaT cell receptor; anti-CD19 plus anti-CD5; anti-CD4 plus anti-CD45RA; or anti-CD8 plus anti-CD11b. Triple staining was performed with fluorescent antibodies against CD4, interferon gamma, and interleukin-4. Both staining protocols were followed by flow cytometric analysis. RESULTS: Acute hepatitis patients had a high proportion of CD3+ T cells with increased CD4+CD45RA-T helper and CD8+CD11b- cytotoxic T cells. Compared to this group, chronic hepatitis patients showed a decrease in CD4+ cells and an increase in CD19+ B cells and interleukin-4-producing Th2 cells. Cirrhotic patients showed decreased circulating lymphocytes and a low proportion of CD8+ cells accompanied by a decrease in cytotoxic T cells. Furthermore, their lymphocyte profiles showed decreases in primordial lymphocyte subpopulations (T cells with gammasigmaT cell receptors and B cells with CD5). CONCLUSIONS: Although the same pathogenic agent was involved, immune dynamics differed greatly according to duration of viral infection.

Recurrent hepatocellular carcinoma with rapid growth after cardiac surgery.

A 65-year-old man diagnosed with hepatitis C virus-positive hepatitis and severe valvular heart disease was scheduled to undergo cardiac valve replacement. We then found hepatocellular carcinoma in the liver. Because of his severe cardiac dysfunction, we treated him surgically with radiofrequency ablation for the hepatocellular carcinoma only. We continued medical treatment of the heart disease. He hoped to undergo with cardiac surgery one year later for the cardiac dysfunction. There was no evidence of tumor recurrence. We informed him that cardiac surgery requiring extracorporeal circulation might lead to tumor recurrence. He agreed to cardiac valve replacement, and the surgery was successful. Recurrent hepatocellular carcinoma was found in the liver 1 month after the surgery. Over the next month, the tumor progressed rapidly, showing portal vein thrombi. We believe the use of extracorporeal circulation in particular triggered the rapid growth of the recurrent hepatocellular carcinoma. This is the first report of a recurrent hepatocellular carcinoma associated with hepatitis C virus that progressed extensively after cardiac surgery.

Mutations of the interferon sensitivity-determining region (ISDR) correlate with the complexity of hypervariable region (HVR)-1 in the Japanese variant of hepatitis C virus (HCV) type 1b.

Hepatitis C virus (HCV) genotype 1b comprises mainly two subtypes in Japan, each named for its geographic prevalence (Japan-specific, J type; worldwide, W type). Because the newly identified subtypes have not been fully characterized, the present study directed this issue from virological viewpoints such as hypervariable region (HVR)-1 as well as interferon (IFN) sensitivity-determining region (ISDR). Fifty chronic hepatitis patients with HCV 1b (31 men and 19 women; mean age 50.5 years) were enrolled, and J/W type was determined according to envelope 1 (E1) sequence as described previously (23 J type and 27 W type). Correlations between age, number of HVR-1 clones, HVR-1 diversity, and ISDR mutations were analyzed in J and W type patients independently. In addition, the sequences of the three HCV regions obtained for the determination of the above genetic factors were studied phylogenetically. The number of HVR-1 clones was significantly higher for J type in comparison with W type (P = 0.044). In the J type-infected patients, the ISDR mutation number was correlated inversely with HVR-1 clone number (P = 0.0001, r = -0.734) and HVR-1 diversity (P = 0.0001, r = -0.722). However, this correlation was not observed in the W type patients. W type patients showed a significant correlation between age and HVR-1 clone number (P = 0.015, r = 0.462). Phylogenetic study revealed that the nonstructural (NS) 5A sequence, which is obtained for ISDR type determination, can distinguish between J and W types. The inverse correlation in J type patients between ISDR mutations and HVR-1 complexity may explain the usefulness of the ISDR for prediction of IFN response only in Japanese patients. This suggests that the ISDR is not directly related to IFN responsiveness, but the degree of HVR-1 complexity may be more important.

Effects of liver failure on branched-chain alpha-keto acid dehydrogenase complex in rat liver and muscle: comparison between acute and chronic liver failure.

BACKGROUND/AIMS: Branched-chain alpha-keto acid dehydrogenase (BCKDH) complex catalyses the committed step in the branched-chain amino acid (BCAA) catabolic pathway. In many cases of liver failure, the serum BCAAs/aromatic amino acids ratio (Fisher's ratio) decreases, and BCAAs have been administered to patients with liver failure to correct this ratio. We conducted an animal study to examine whether the effects on hepatic BCKDH complex differ between acute liver failure (ALF) and chronic liver failure (CLF). METHODS: ALF and CLF was induced in rats by a single high-dose injection and 21 weeks of repeated low-dose injections of carbon tetrachloride, respectively. Plasma BCAA and branched-chain alpha-keto acid (BCKA) levels, and activities and protein amounts of hepatic BCKDH complex and kinase were measured. RESULTS: ALF was characterized by elevated plasma BCAA and BCKA levels and decreased hepatic BCKDH activity. CLF was characterized by decreased plasma BCAA and BCKA levels and increased hepatic BCKDH activity. This increase in BCKDH activity in CLF was associated with the decreased BCKDH kinase, which is responsible for the BCKDH inactivation. CONCLUSIONS: The results obtained in the present study suggest that BCAA catabolism is suppressed in ALF and increased in CLF.

Comparison of contrast-enhanced harmonic ultrasonography and power Doppler ultrasonography for depicting vascularity of hepatocellular carcinoma identified by angiography-assisted CT.

The sensitivity of contrast-enhanced harmonic gray scale imaging was compared with that of power Doppler sonography in depicting tumor vascularity and contrast enhancement of hepatocellular carcinoma (HCC) identified by angiography-assisted computed tomography (CT). One hundred thirty-nine classic HCC nodules (108 patients), each appearing as a high-attenuation mass on early-phase CT during hepatic arteriography and as a perfusion defect on CT during arterial portography, were evaluated. Vascular findings and contrast enhancement patterns were evaluated by contrast-enhanced harmonic gray scale imaging. Arterial pulsatile flow into each nodule was checked by power Doppler sonography. Sensitivity of each modality for depicting tumor vascularity was examined by angiography-assisted CT findings as the gold standard. The sensitivity of contrast-enhanced harmonic gray scale imaging (134/139, 96.4%) was significantly greater than that of power Doppler sonography (96/139, 69.1%) (P<0.05). Twenty-six of twenty-seven nodules (96.3%) in the lateral segment, in which motion artifact is likely, were enhanced by contrast-enhanced harmonic gray scale imaging. Contrast-enhanced harmonic gray scale imaging is more sensitive than power Doppler sonography and is a noninvasive method that can be used as effectively as angiography-assisted CT to evaluate tumor vessels and contrast enhancement of HCC.

Characteristics and prognosis of hepatocellular carcinoma detected in sustained responders to interferon therapy for chronic hepatitis C.

Interferon (IFN) therapy allows the eradication of hepatitis C virus (HCV) in some part of patients with chronic hepatitis C which is major cause of hepatocellular carcinoma (HCC). To clarify characteristics and prognoses of HCC detected in these patients (sustained responders to IFN), we compared HCC in sustained responders with HCC detected in patients without a sustained response (non-sustained responders). Characteristics and prognoses were compared in nine cases of HCC detected in sustained responders after IFN therapy and 61 cases of HCC detected in non-sustained responders at one of five our institutions. HCC in sustained responders often were larger (P=0.0051), less differentiated tumor (P=0.0084) than HCC in non-sustained responders when it was detected. No differences were observed in overall survival rate between sustained responders and non-sustained responders, but disease-free survival was higher in cases of HCC in sustained responders (P=0.0494). HCC detected in sustained responders often appear more advanced when detected than HCC in non-sustained responders, but recurrence seems to be less frequent when the initial HCC is treated sufficiently.

A novel subtype of GB virus C/hepatitis G virus genotype 1 detected uniquely in patients with hemophilia in Japan.

GB virus C (GBV-C) or hepatitis G virus (HGV) has been transmitted to Japanese patients with hemophilia through the frequent use of unheated blood products. Sequence analysis showed that most of the viruses isolated from these patients belonged to GBV-C/HGV genotype 1, which is usually found in persons from Africa. This may point to the origin of this virus in Japanese patients with hemophilia. The phylogeny of 11 GBV-C/HGV isolates from Japanese patients with hemophilia was investigated by a detailed analysis with a fragment spanning from the 5' non-coding region to part of the E1 gene. Except for one that belonged to the genotype 3 cluster, all isolates were GBV-C/HGV type 1. Five main clades exist within the GBV-C/HGV genotype 1 sequences. These isolates are grouped in 2 defined clades. Three of the isolates are clustered in subtype 1c clade whereas the other 7 strains formed a new statistically well-supported monophyletic group (named subtype 1e). Our results suggest that GBV-C/HGV type 1 can at present be classified into at least 5 clades and in this group a majority of Japanese patients with hemophilia was infected with a GBV-C/HGV of a unique and newly described subtype within genotype 1.

Prevalence and characterization of hepatitis C virus genotype 4 in Japanese hepatitis C carriers.

Hepatitis C virus (HCV) can be classified into six major genotypes, the prevalences of which differ around the world. In Japan, the main genotypes are HCV 1 and HCV 2; others are found only rarely. Little is known about the prevalence in Japan of HCV genotype 4 which, is found frequently in North and Central Africa and the Middle East. Thus, we conducted a study to clarify distribution of HCV genotype 4 and the clinical demographics of patients with HCV genotype 4 in Japan. We examined HCV genotypes in 899 Japanese individuals with HCV viremia living in Aichi Prefecture, including 63 hemophiliacs. Four patients (0.4%) were infected with HCV genotype 4. All four of these patients were male hemophiliacs who had received clotting factors from foreign countries. Three patients were co-infected with human immunodeficiency virus (HIV); none were co-infected with GB virus-C/hepatitis G virus. Phylogenetic analysis of the El region indicated that all four patients were infected with subtype 4a. This subtype is related genetically to a subtype previously reported in Japanese and Italian hemophiliacs. HCV genotype 4 is indeed rare in Japan and may be detected only among hemophiliacs who have received inactivated clotting factor concentrates from foreign countries.

Toll-like receptor 2 contributes to liver injury by Salmonella infection through Fas ligand expression on NKT cells in mice.

BACKGROUND & AIMS: Toll-like receptors (TLRs) for bacterial constitutes are expressed not only by phagocytes but also by some subsets of T cells. We previously reported that natural killer T cells (NKT cells) play an important role in liver injury induced by Salmonella infection. In the present study, we investigated whether TLRs on NKT cells are involved in Salmonella-induced liver injury. METHODS: Gene expression of TLR2 was examined in sorted natural killer, NKT, and T cells from livers of naive mice by the reverse-transcription polymerase chain reaction method. Serum alanine aminotransferase level and FasL expression on liver lymphocytes were examined in TLR2-deficient (TLR2(-/-)) and FasL-deficient gld/gld mice before and after intraperitoneal inoculation of Salmonella choleraesuis 31N-1 using an enzyme-linked immunosorbent assay and flow cytometry. RESULTS: TLR2 gene was abundantly expressed by NKT cells freshly isolated from naive mice. FasL expression on liver NKT cells increased in TLR2(+/-) mice but not in TLR2(-/-) mice after Salmonella infection. Serum alanine aminotransferase level was significantly lower in the TLR2(-/-) and gld/gld mice than in the control mice after infection. CONCLUSIONS: TLR2 may contribute to liver injury induced by Salmonella infection via FasL induction on liver NKT cells.

Evaluation of a new assay for hepatitis C virus genotyping and viral load determination in patients with chronic hepatitis C.

A new method for hepatitis C virus (HCV) genotyping that analyzes products generated with the HCV Amplicor Monitor Test has been developed. One hundred and sixty-two Japanese patients with chronic hepatitis C, including 59 patients with hemophilia, were tested for HCV genotypes and viral loads with this new test, and the results were compared with those of a genotyping assay that involved direct sequencing of the E1 region. HCV genotypes and viral loads were also compared between patients with and without hemophilia. There were no discrepancies between the two methods in determining genotypes 2a, 2b, and 3a. However, two patients infected with 1a were mistyped as 1b with the new assay. One patient not classified by this assay was genotype 4. Genotypes found in patients without hemophilia were 1b, 2a, and 2b. Genotypes 1a, 3a, and 4, which were minor variants in Japan, were detected only in patients with hemophilia. In addition, J type, which is a subtype of 1b that originated in Japan, was found at low frequency in hemophiliacs. Thus, the HCV genotypes in patients with hemophilia are likely to be of foreign origin. Overall, this new assay was accurate except for genotype 1a and 4, and allowed simultaneous assessment of genotype and viral load.

[Impact of HIV on HCV, GBV-C/HGV, and HBV infection].

To investigate influence of HIV co-infection on HCV, we examined 58 Japanese hemophiliacs with chronic hepatitis C(CHC) including 25 patients co-infected with HIV. HCV RNA levels and liver function were not affected statistically by the presence of HIV. Further studies are necessary to evaluate the impact of HIV on the prognosis of CHC in hemophiliacs. We were the first to report a beneficial effect of GBV-C/HGV infection on the course of HIV disease and many studies were confirmed our results. Discussions of these important issues such as impact of HIV on GBV-C/HGV and HBV have been performed in this paper.

Forced expression of NESH suppresses motility and metastatic dissemination of malignant cells.

To characterize the function of a novel Src homology 3 (SH3) adapter protein termed NESH, we have established transfectants stably expressing NESH. We observed that every clone of NESH transfectants caused a marked reduction in motility, although the clones exhibited no significant differences in intrinsic cell growth compared with the control cells in vitro. The NESH transfectants also exhibited significant reduction in tumor metastatic potential in vivo. We found that NESH expression is frequently lost in invasive cancer cell lines despite its ubiquitous expression in normal tissues. The SH3 domain of NESH seems to interact with p21-activated kinase (PAK), which is involved in regulation of cell motility. Furthermore, a significant decrease in PAK phosphorylation at (402)Thr was found in NESH transfectants. Taken together, these results suggest that NESH inhibits ectopic metastasis of tumor cells as well as cell migration through interaction with intracellular molecules such as PAK that are essential for cell motility.