[Case of chronic type C hepatitis complicated with idiopathic thrombocytopenic purpura that was successfully treated by interferon therapy].

We report a case of chronic hepatitis C complicated with idiopathic thrombocytopenic purpura (ITP), successfully treated with interferon (IFN) beta. A 65-year-old woman was admitted to our hospital for the treatment of chronic hepatitis C with IFN beta. ITP was also diagnosed because of the presence of platelet associated IgG and the findings of bone marrow examination. We started IFN therapy, which resulted in normalization of transaminases, complete HCV eradication, and increased number of platelet.

What can be revealed by extending the sensitivity of HBsAg detection to below the present limit?

BACKGROUND/AIMS: We investigated what can be revealed by extending the sensitivity of HBsAg detection to below the present limit. METHODS: We examined the sensitivity of this immunoassay in comparison with real-time PCR detection of HBV DNA using serially diluted sera from HBV carriers. Low HBsAg was measured in 210 healthy volunteers and 368 patients with non-B chronic liver diseases who were negative for HBsAg by a standard EIA method. RESULTS: The radical immunoassay was able to detect HBsAg at a concentration of 0.025 ng/ml. Low HBsAg was positive in 6 of 210 normal volunteers (2.86%), 5 of 65 non-B, non-C cirrhosis patients (7.69%), 6 of 62 non-B, non-C hepatocellular carcinoma patients (9.68%: p=0.04 vs. volunteers), 12 of 134 chronic hepatitis C patients (8.96%: p<0.02 vs. volunteers), and 11 of 107 hepatocellular carcinoma patients complicated by chronic hepatitis C (10.28%: p<0.008 vs. volunteers). Although no HBV DNA was positive in healthy volunteers, 9 patients with non-B chronic liver diseases were positive for HBV DNA by real-time PCR analysis. CONCLUSIONS: Increasing the sensitivity of HBsAg detection to below the present limit has revealed that infection with HBV, including occult HBV, is far more endemic than suspected previously.

[Two cases of pyogenic spondylitis with chronic hepatitis C during combination therapy of interferon alfa and ribavirin].

This report describes our experience with two cases of pyogenic spondylitis with chronic hepatitis C during combination therapy of interferon alfa and ribavirin. The first patient, a 59-year-old man, was treated conservatively and improved, but the second patient, a 69-year-old woman, was not improved by conservative therapy and reconstructive operation was performed. The combination therapy of interferon alfa and ribavirin has a high risk of severe infectious diseases as side effects. CT scan and MRI are recommended immediately to diagnose pyogenic spondylitis, when patients has pyrexia and lumbago with laboratory data suspected inflammation during interferon therapy.

Risk of hepatocellular carcinoma and secondary structure of hepatitis C virus (HCV) NS3 protein amino-terminus, in patients infected with HCV subtype 1b.

We conducted a retrospective study of 65 patients with chronic hepatitis C, to determine whether the secondary structure of the amino-terminal 120 residues of the hepatitis C virus (HCV) NS3 protein is associated with an increased risk of development of hepatocellular carcinoma (HCC). The cumulative incidence of HCC was highest among patients infected with group B HCV-1b, wherein the risk of HCC significantly increased compared with that among patients infected with group A (hazard ratio, 4.95 [95% CI, 1.43-17.11]) after adjustment for age and histological stage. This HCV-1b grouping may be a useful marker for detecting the risk of development of HCC.

Serum levels of stem cell factor and thrombopoietin are markedly decreased in fulminant hepatic failure patients with a poor prognosis.

BACKGROUND AND AIM: Hematopoietic growth factors including stem cell factor (SCF), thrombopoietin (TPO) and granulocyte colony stimulating factor (G-CSF) have a potential role in inducing bone marrow hematopoietic stem cells to move into the circulation, and the association of these factors with liver regeneration has received a lot of attention recently. The aim of this study was to determine the serum levels of such factors in patients with acute liver injury. METHODS: The subjects were 25 patients with acute hepatitis (AH) who had a favorable prognosis and 26 patients with fulminant hepatitis (FH), of whom 11 were alive and 15 had died. Sixty-six healthy subjects matched for age and sex served as controls. Serum samples were collected before treatment, and the levels of SCF, TPO and G-CSF were measured using enzyme-linked immunosorbant assays. RESULTS: The levels of SCF and TPO were significantly lower in FH patients than in AH patients and the controls, and were also significantly lower in the FH patients who died, compared to the surviving patients. The G-CSF levels did not differ among them. CONCLUSIONS: These results suggest that low serum levels of SCF and TPO may be linked to poor prognosis in patients with severe liver injury.

Separate analysis of asialoglycoprotein receptors in the right and left hepatic lobes using (99m)Tc-GSA SPECT in patients with acute hepatic damage.

The asialoglycoprotein receptor (ASGPR) is abundantly expressed on the sinusoidal surfaces of hepatocytes. However, regional expression and clinical significance of the ASGPR in acute hepatic damage is presently unknown. Our aim was to clarify the clinical significance of the regional expression of ASGPR in human livers with acute hepatitis (AH) and fulminant hepatic failure (FHF). Eighteen volunteers, 42 patients with AH and 10 with FHF were studied using a newly developed, conventional (99m)Tc-GSA SPECT analysis. Using Cantlie's line as a guide, ASGPR expression was analyzed separately in the right and left hepatic lobes, as well as in the whole liver, using novel indices (the liver uptake ratio [LUR] and liver uptake density [LUD], which reflect the amount and density of ASGPRs in the liver, respectively). Mean LUR and LUD values for the whole liver and the right and left lobes decreased in accordance with the severity of acute hepatic damage. In the FHF group, the reduction in LUR and LUD values in the right lobes was more significant than in the left lobes. The LUR and LUD values for the whole liver correlated well with hepatic functional reserve and total bilirubin levels. The right LUR and LUD values in particular correlated well with these parameters. A time-course observation of 13 patients with either AH or FHF revealed that the expression of ASGPRs in the right lobe recovered faster than in the left. We first evaluated the regional expression of AGSPRs by (99m)Tc-GSA SPECT analysis in both AH and FHF patients, which is a clinically useful and reliable indicator for assessing the severity of regional hepatic damage and evaluating regional liver regeneration.

Characteristics of rat bone marrow cells differentiated into a liver cell lineage and dynamics of the transplanted cells in the injured liver.

BACKGROUND: Bone marrow cells (BMCs) have been shown to differentiate into a liver cell lineage, but little is known about their dynamics following transplantation. BMCs were cultured to investigate the expression of liver-specific genes in vitro and transplanted into in vivo liver-injury models to elucidate their dynamics in the liver. METHODS: The mRNA expression of various liver-specific genes in BMCs cocultured with hepatocytes was analyzed using reverse transcription-polymerase chain reaction. BMCs from transgenic rats expressing green fiuorescent protein were transplanted into the spleen of rat liver-injury models induced with 2-acetylaminofiuorene (2-AAF) or carbon tetrachloride (CCl4). BMCs were also transplanted directly into livers treated with CCl4 to determine which route is better for transplantation. RESULTS: BMCs differentiated into a liver cell lineage in vitro and expressed mRNAs consistent with mature hepatocytes, including albumin. The transplanted BMCs were found in the liver in the CCl4-induced injury model, but not in the 2-AAF-induced model. The hepatocyte growth factor and fibroblast growth factor mRNA levels in the liver were significantly higher in the CCl4-induced model than in the 2-AAF-induced model. Migration of BMCs to the liver was more effective following injection into the liver, rather than into the spleen. CONCLUSIONS: Cultured BMCs differentiated into a liver cell lineage are a potential source for cell transplantation. Transplantation is successful in the severely injured liver with a high level of expression of mRNAs for growth factors. Injection of BMCs directly into the liver is the preferred route of administration.

Reduction of serum ghrelin concentration during interferon-alpha therapy in patients with chronic hepatitis C.

The efficacy of interferon (IFN) therapy for chronic hepatitis C is dependent on compliance. Anorexia is an important adverse effect in determining compliance. To clarify the mechanisms underlying anorexia, the level of ghrelin was determined during therapy. Fourteen patients with chronic hepatitis C received IFN-alpha2b with or without ribavirin (Rib+ or Rib- group; n=7 in each group) for 24 weeks. Serum ghrelin concentrations and body weight were determined before, 2 and 24 weeks after initiation of therapy. Serum ghrelin concentrations and body weight significantly decreased 2 weeks after initiation of therapy (P=0.0008 and 0.0062, respectively), and then returned to the level before therapy. The Deltaghrelin concentration correlated with Deltabody weight after 2 weeks (r=0.726, P=0.023). Percentage reduction of serum ghrelin was significantly higher in the Rib+ group than in the Rib- group (P=0.046). Percentage reduction in body weight tended to be higher in the Rib+ group (P=0.057). IFN-alpha2b therapy causes short-term reduction of serum ghrelin and body weight, and this may occur to a greater extent with combination therapy. Reduction of serum ghrelin might contribute partly to anorexia, leading to weight loss.

NAD(P)H oxidase plays a crucial role in PDGF-induced proliferation of hepatic stellate cells.

The proliferation of hepatic stellate cells (HSCs) is a critical step in hepatic fibrogenesis. Platelet-derived growth factor (PDGF) is the most potent mitogen for HSCs. We investigated the role of nonphagocytic NAD(P)H oxidase-derived reactive oxygen species (ROS) in PDGF-induced HSC proliferation. The human HSC line, LI-90 cells, murine primary-cultured HSCs, and PDGF-BB were used in this study. We examined the mechanism of PDGF-BB-induced HSC proliferation in relation to the role of a ROS scavenger and diphenylene iodonium, an inhibitor of NAD(P)H oxidase. We also measured ROS production with the aid of chemiluminescence. We showed that PDGF-BB induced proliferation of HSCs through the intracellular production of ROS. We also demonstrated that HSCs expressed key components of nonphagocytic NAD(P)H oxidase (p22phox, gp91phox, p47phox, and p67phox) at both the messenger RNA and protein levels. Diphenylene iodonium suppressed PDGF-BB-induced ROS production and HSC proliferation. Coincubation of H2O2 and PDGF-BB restored the proliferation of HSCs that was inhibited by diphenylene iodonium pretreatment. Phosphorylation of the mitogen-activated protein kinase (MAPK) family constitutes a signal transduction pathway of cell proliferation. Our data demonstrate that NAD(P)H oxidase-derived ROS induce HSC proliferation mainly through the phosphorylation of p38 MAPK. Moreover, an in vivo hepatic fibrosis model also supported the critical role of NAD(P)H oxidase in the activation and proliferation of HSCs. In conclusion, NAD(P)H oxidase is expressed in HSCs and produces ROS via activation of NAD(P)H oxidase in response to PDGF-BB. ROS further induce HSC proliferation through the phosphorylation of p38 MAPK.

Differentiation of rat bone marrow cells cultured on artificial basement membrane containing extracellular matrix into a liver cell lineage.

BACKGROUND/AIMS: Bone marrow (BM) cells have been shown to be capable of differentiating into a liver cell lineage in vitro. However, their differentiation and proliferation is poor, and the cell characteristics are poorly understood. METHODS: We cultured rat BM cells on an artificial basement membrane containing extracellular matrix (ECM) with hepatocyte growth factor (HGF). The expression of mRNA for liver-specific genes was analyzed by reverse transcription PCR. The expression of albumin and Musashi-1 by cultured cells was analyzed using a fluorescence-activated cell sorter (FACS). The proportions of albumin-positive cells when culture was performed with different concentrations of HGF were analyzed by FACS. RESULTS: On culture day 21, polygonal cells proliferated and formed cell colonies. These cells expressed mRNA for all the liver-specific genes analyzed, and showed heterogeneous differentiation, some cells expressing albumin, others expressing Musashi-1. Albumin-positive differentiated cells were large and rich in intracellular structures, while Musashi-1-positive undifferentiated cells had the opposite characteristics. Culturing cells with higher concentrations of HGF induced an increased proportion of albumin-positive cells. CONCLUSIONS: The results suggest that cell culture on an ECM with a high concentration of HGF increases the extent to which BM cells differentiate into a liver cell lineage and proliferate in vitro.

Expression of Notch signalling markers in bone marrow cells that differentiate into a liver cell lineage in a rat transplant model.

Notch signalling pathway plays an important role in cell differentiation. To investigate the implications of Notch signalling in the differentiation of rat bone marrow (BM) cells into a liver cell lineage, we used cultured BM cells to examine the mRNA expression of Musashi-1, which positively regulates Notch signalling, and made a transplant model to examine the protein expression of Notch signalling markers. For the in vivo experiment, BM cells were collected from transgenic rats expressing green fluorescence protein (GFP) and transplanted into the spleens of recipient rats, in which liver damage had been induced with carbon tetrachloride. The expression of Notch receptor 1 (Notch-1), Jagged-1 and Musashi-1, in the transplanted GFP-positive BM cells was investigated by immunohistochemistry. The expression of the liver-specific proteins, alpha-fetoprotein and cytokeratin19 was also investigated. Musashi-1 mRNA became detectable in the BM cells on culture day 7 in vitro. After transplantation, GFP-positive BM cells were observed in the portal areas of the recipient's livers. Notch-1, Jagged-1, Musashi-1, alpha-fetoprotein and cytokeratin19 were all expressed in the transplanted BM cells. These results suggest that the Notch signalling pathway plays a role in the differentiation of BM cells into a liver cell lineage.

Transmission of hepatitis C virus quasispecies between human adults.

To elucidate how hepatitis C virus (HCV) with multiple variants (quasispecies) is transmitted and adapts to the host during infection, we compared nucleotide and deduced amino acid sequences from hypervariable region1 (HVR1) of the E2 gene of HCV between a donor and a recipient who developed hepatitis after a needlestick accident. Thirty clones from each subject were sequenced after PCR amplification, cloning, and purification of plasmid DNA from single colonies of transformed bacteria. Genetic analysis revealed that the recipient's viral sequences were much less diverse than the donor's. We found a single predominant HCV HVR1 clone of the recipient in 22/30 isolates with the same amino acid sequence, and mimic clones in 8/30 isolates with only one amino acid substitution. These were all absent in the donor, who had 21 highly diverse sequences. Phylogenetic analysis of virus E1/E2 gene sequences showed that the recipient's unique sequences were related to the population of variants from the donor, in whom one isolate had 96% similarity to the recipient's predominant amino acid sequence. These results suggest that a minor subset of the donor's HCV variants is selectively transmitted to the recipient, and that the selection determines the predominant variant in the new host.

Genetic variations in humans associated with differences in the course of hepatitis C.

The outcome of hepatitis C virus (HCV) infection varies among individuals, but the genetic factors involved remain unknown. We conducted a population-based association study in which 238 Japanese individuals positive for anti-HCV antibody were genotyped for 269 single nucleotide polymorphisms (SNPs) in 103 candidate genes that might influence the course of infection. Altogether, 50 SNPs in 32 genes were listed. Genetic polymorphisms in IL4, IL8RB, IL10RA, PRL, ADA, NFKB1, GRAP2, CABIN1, IFNAR2, IFI27, IFI41, TNFRSF1A, ALDOB, AP1B1, SULT2B1, EGF, EGFR, TGFB1, LTBP2, and CD4 were associated with persistent viremia (P < 0.05), whereas those in IL1B, IL1RL1, IL2RB, IL12RB1, IL18R1, STAT5A, GRAP2, CABIN1, IFNAR1, Mx1, BMP8, FGL1, LTBP2, CD34, and CD80 were associated with different serum alanine aminotransferase levels in HCV carriers (P < 0.05). The sorted genes allow us to draw novel hypotheses for future studies of HCV infection to ultimately identify bona fide genes and their variations.

Separate analysis of asialoglycoprotein receptors in the right and left hepatic lobes using Tc-GSA SPECT.

The asialoglycoprotein receptor (ASGPR) is abundantly expressed on the sinusoidal surfaces of hepatocytes. We aimed to clarify the clinical significance of the regional distribution of ASGPRs in the human liver, especially in chronic viral hepatitis. Eighteen volunteers, 34 patients with chronic hepatitis, and 33 patients with cirrhosis (11/Child-Pugh A, 11/Child-Pugh B, 11/Child-Pugh C) were studied using a newly developed, conventional technetium-99m-diethylenetriaminepentaacetic acid-galactosyl human serum albumin ((99m)Tc-GSA), single photon emission computed tomography (SPECT) method. Using Cantlie's line as a guide, ASGPR dynamics were analyzed separately in the right and left lobes, as well as in the whole liver, using novel indices (the liver uptake ratio [LUR] and the liver uptake density [LUD], which reflect the amount and density of ASGPRs in the liver, respectively). Mean LUR and LUD values for the whole liver and the right and left lobes decreased with increasing progression of chronic viral hepatitis. The LUR for the whole liver correlated well with parameters measuring the hepatic functional reserve and the platelet count. The right LUR correlated particularly well with conventional liver function tests, and comparison of the right LUD with histologic findings showed that it was a good indicator of periportal and/or bridging necrosis and fibrosis. In conclusion, our (99m)Tc-GSA SPECT method was clinically useful in evaluating regional hepatic function and the progression of chronic viral hepatitis using dynamic changes in ASGPRs.

Possible contribution of circulating interleukin-10 (IL-10) to anti-tumor immunity and prognosis in patients with unresectable hepatocellular carcinoma.

OBJECTIVES: Interleukin-10 (IL-10) has been implicated in immune deficiency in patients with cancer. The relationship of this cytokine as measured in serum to anti-tumor immunity and prognosis was investigated in unresectable hepatocellular carcinoma. METHODS: This study consisted of 74 consecutive patients with unresectable hepatocellular carcinoma (median age, 65 years). Forty-four healthy age-matched subjects and 32 patients with cirrhosis but no carcinoma served as controls. Patients with hepatocellular carcinoma were divided into those with serum IL-10 concentrations above (high group, n=39) or below (low group, n=35) 10pg/ml. RESULTS: Age, gender, Child-Pugh grade, and tumor stage distributions were similar in high and low groups. The patients of high group showed lower in lymphokine-activated killer (LAK) and natural killer (NK) activities than those of low group (P<0.01 and 0.01, respectively). Serum IL-10 concentration was a significant factor contributing to low activities of LAK and NK by logistic regression analysis (P<0.05 and 0.05, respectively). The high group had a significantly shorter survival (median, 3 months) than low group (median, 12 months; P<0.02, generalized Wilcoxon test). CONCLUSIONS: These data suggest that serum IL-10 concentration is a possible factor contributing to poor prognosis and low anti-tumor immunity in patients with unresectable hepatocellular carcinoma.

Spontaneous elimination of serum hepatitis C virus (HCV) RNA in chronic HCV carriers: a population-based cohort study.

The natural course of hepatitis C virus (HCV) infection has not been fully elucidated. To investigate whether HCV is spontaneously eliminated in chronic carriers, a long-term population-based cohort study was conducted on 435 chronic HCV carriers. Individual characteristics, serum HCV RNA, and liver function tests were analyzed, and ultra sonography (US) was performed in all subjects. Subjects were followed up for 7.2 +/- 2.4 years (mean +/- SD). Serum HCV RNA was spontaneously eliminated in 16/435 (3.7%) individuals during this period; thus, the incidence of spontaneous elimination of serum HCV RNA was 0.5%/year/person. Multivariate analysis revealed that both a low value of ZTT and no US finding of chronic liver disease were associated with spontaneous viral elimination in HCV carriers. Three of these 16 individuals had chronic hepatitis, and 13 of them had normal ALT levels. When the neutralization of binding (NOB) assay that evaluates inhibition of the HCV envelope-2 protein binding to human cells was examined using sera from these 16 individuals, the NOB antibody was detected in only 3 cases with chronic hepatitis. These results suggest that serum HCV RNA is spontaneously eliminated in chronic HCV carriers in a population, and that the development of NOB antibody is associated with a natural resolution of chronic hepatitis in the minority of them.

Differentiation of bone marrow cells into cells that express liver-specific genes in vitro: implication of the Notch signals in differentiation.

Bone marrow (BM) stem cells have been shown to differentiate into liver cells. It remains difficult to sort and culture BM stem cells, and the gene expression of liver-specific proteins in these cells has not been fully investigated. We used a negative selective magnetic cell separation system to obtain stem cell-enriched BM cells. The cells obtained were cultured with hepatocytes or with hepatocyte growth factor (HGF), and the differentiation of BM cells into cells expressing liver-specific genes, hepatocyte nuclear factor (HNF) 1alpha, cytokeratin (CK) 8, alpha-fetoprotein (AFP), and albumin was investigated by the reverse transcription-polymerase chain reaction. We also investigated the gene expressions of Notch receptor-1 (Notch-1) and its ligand Jagged-1 in BM cell differentiation. Sorted BM cells showed positive for Sca-1 (Ataxin-1) by immunofluorescence staining. Fluorescence activated cell sorter analysis showed that 32.6% of sorted BM cells had a high level of expression of the hematopoietic stem cell marker CD90 (Thy-1). When cultured with hepatocytes, these cells expressed the liver-specific genes HNF1alpha and CK8 on culture day 3, AFP and albumin on culture day 7. When cultured with HGF (20ng/ml), the cells expressed HNF1alpha on day 3 and CK8 on day 7. Gene expressions of Notch-1 and Jagged-1 were detected in cultured BM cells on day 3. These results suggest that the negative selective magnetic cell separation system is useful for the rapid preparation of stem cell-enriched BM cells, and that the Notch signaling pathway plays a role in BM cell differentiation into a hepatocyte lineage in vitro.