Kazuo Okumoto

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.

Possible contribution of circulating transforming growth factor-beta1 to immunity and prognosis in unresectable hepatocellular carcinoma.

Transforming growth factor‐β1 (TGF‐β1) has been implicated in tumor progression. The relationship of this cytokine as measured in plasma to anti‐tumor immunity and prognosis was investigated.

Potential therapeutic application of intravenous autologous bone marrow infusion in patients with alcoholic liver cirrhosis.

The present study was conducted to evaluate the application and efficacy of autologous bone marrow infusion (ABMi) for improvement of liver function in patients with alcoholic liver cirrhosis (ALC). Five subjects and 5 control patients with ALC who had abstained from alcohol intake for 24 weeks before the study were enrolled. Autologous bone marrow cells were washed and injected intravenously, and the changes in serum liver function parameters, and the level of the type IV collagen 7S domain as a marker of fibrosis, were monitored for 24 weeks. The distribution of activated bone marrow was assessed by indium-111-chloride bone marrow scintigraphy. The number of cells infused was 8.0±7.3×10(9) (mean±standard error). The serum levels of albumin and total protein and the prothrombin time were significantly higher during the follow-up period after ABMi than during the observation period in treated patients, whereas no such changes were observed in the controls. In the patients who received ABMi, the Child-Pugh score decreased in all 3 who were classified as class B; the serum levels of type IV collagen 7S domain improved in 4 of the 5 patients; and bone marrow scintigraphy demonstrated an increase of indium-111-chloride uptake in 3 of the 4 patients tested. ABMi for patients with ALC helps improve liver function parameters in comparison with observation during abstinence and ameliorates the degree of fibrosis in terms of serum markers and bone marrow activation in most cases.

Impact of metabolic syndrome on elevated serum alanine aminotransferase levels in the Japanese population.

Measurement of the serum alanine aminotransferase (ALT) level is used as an initial test for detection of liver diseases, and recent studies have also highlighted its potential value as a measure of overall health and survival as a marker of an increased risk of metabolic disorder. This study was designed to clarify the prevalence of elevated ALT levels in the Japanese population and to assess factors associated with ALT elevation. The subjects were 2165 individuals aged 40 to 85 years who participated in a Japanese community-based study referred to as the Takahata Study. Serum ALT levels and factors associated with ALT elevation were investigated. Among 2087 subjects who were negative for hepatitis B and C, the rates of elevated ALT greater than 30 U/L in men and greater than 25 U/L in women were 217 (22.7%) of 957 and 239 (21.2%) of 1130, respectively. These ALT cutoff levels had a specificity of more than 80% for exclusion of subjects with none or 1 of 3 metabolic risk factors: hypertension, lipid metabolism abnormality, and hyperglycemia. Multivariate analysis revealed 5 factors with a significant association with ALT elevation in men (n = 957): high gamma-glutamyltranspeptidase, low adiponectin, high low-density lipoprotein cholesterol, high body mass index, and high homeostasis model assessment insulin resistance index. Similarly, 4 factors were significantly associated with ALT elevation in women (n = 1130): high gamma-glutamyltranspeptidase, low adiponectin, high body mass index, and high homeostasis model assessment insulin resistance index. These results suggest that elevated ALT levels in the Japanese population older than 40 years have a strong association with metabolic syndrome-related features including obesity and insulin resistance.

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

BackgroundBone 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.MethodsThe 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.ResultsBMCs 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.ConclusionsCultured 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.

Dynamics of serum metabolites in patients with chronic hepatitis C receiving pegylated interferon plus ribavirin: A metabolomics analysis

OBJECTIVES Serum samples from patients with chronic hepatitis C were subjected to metabolomics analysis to clarify the pretreatment characteristics of their metabolites and also changes in specific metabolites resulting from antiviral therapy with pegylated interferon plus ribavirin (PegIFN/RBV). MATERIALS/METHODS The serum levels of low-molecular-weight metabolites in the twenty patients before and 24weeks after completion of PegIFN/RBV therapy were analyzed using capillary electrophoresis and liquid chromatography-mass spectrometry. RESULTS Ten patients showed a non-virological response (NVR) and 10 achieved a sustained virological response (SVR) with eradication of viremia. The pretreatment levels of tryptophan were significantly higher in the patients of SVR than in those of NVR (p=0.010). The area under the curve (AUC) value of tryptophan calculated from the receiver operating characteristic (ROC) curve for discriminating SVR from NVR was 0.84 (95% confidential interval, 0.66-1.02, p=0.010). The ROC curve of multiple logistic regression model incorporating the pretreatment levels of tryptophan and γ-glutamate-arginine showed that the AUC value was highly significant (AUC=0.92, 95% confidential interval, 0.79-1.05, p=0.002). Twenty four weeks after completion of treatment, the levels of γ-glutamyl dipeptides, glutamic acid, 5-oxoproline, glucosamine and methionine sulfoxide were decreased, whereas those of 5-methoxy-3-indoleacetate, glutamine, kynurenine and lysine were increased significantly (p<0.05) in both the NVR and SVR patients. CONCLUSIONS The pretreatment serum levels of certain metabolites including tryptophan are associated with the response to PegIFN/RBV therapy. PegIFN/RBV therapy can ameliorate the oxidative stress responsible for glutathione metabolism.

Relationship between Alcohol Consumption and Serum Adiponectin Levels: The Takahata Study—A Cross-Sectional Study of a Healthy Japanese Population

CONTEXT The relationship between alcohol consumption and serum adiponectin levels has not been fully explored in an Asian population. OBJECTIVE Our goal was to determine whether alcohol consumption is associated with a change in adiponectin levels in a healthy Japanese population. DESIGN This was a cross-sectional study. SETTING Subjects were recruited from participants in a health check-up program. PARTICIPANTS This study included 2932 subjects (1306 men and 1626 women). MAIN OUTCOME MEASURES The effects of total weekly or daily volume of ethanol intake on serum adiponectin levels were evaluated. In addition, the correlation of clinical traits with serum adiponectin levels was examined. A multivariate regression model was used to control for possible confounding factors. RESULTS Alcohol consumption was weakly correlated with decreased serum adiponectin levels in men [Spearmans ordered correlation coefficient (rs=-0.141; P<0.001]; an even weaker correlation was seen in women (rs=-0.055; P=0.025). Multivariate analysis demonstrated that alcohol consumption was independently associated with hypoadiponectinemia. CONCLUSION In contrast to reports from the United States and Europe among White and Black subjects, our study demonstrated an inverse association between alcohol intake and serum adiponectin levels in Asian subjects, suggesting ethnic differences in the effects of alcohol consumption on serum adiponectin levels.

Clinical manifestations of liver injury in patients with anorexia nervosa

The number of Japanese patients with anorexia nervosa (AN) is increasing as society changes. Mild liver injury is a complication of AN in around 30% of cases. In some rare instances, patients present with severe liver injury similar to acute liver failure. However, there are numerous uncertainties over the clinical characteristics of this condition. The objective of the present study was to clarify the clinical characteristics of AN complicated by liver injury and to investigate the factors related to hepatic complications.

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.

Altered Expression of Diacylglycerol Kinase Isozymes in Regenerating Liver

The liver possesses the capacity to restore its function and mass after injury. Liver regeneration is controlled through complicated mechanisms, in which the phosphoinositide (PI) cycle is shown to be activated in hepatocytes. Using a rat partial hepatectomy (PH) model, the authors investigated the expression of the diacylglycerol kinase (DGK) family, a key enzyme in the PI cycle, which metabolizes a lipid second-messenger diacylglycerol (DG). RT-PCR analysis shows that DGKζ and DGKα are the major isozymes in the liver. Results showed that in the process of regeneration, the DGKζ protein, which is detected in the nucleus of a small population of hepatocytes in normal liver, is significantly increased in almost all hepatocytes. However, the mRNA levels remain largely unchanged. Double labeling with bromodeoxyuridine (BrdU), an S phase marker, reveals that DGKζ is expressed independently of DNA synthesis or cell proliferation. However, DGKα protein localizes to the cytoplasm in normal and regenerating livers, but immunoblot analysis reveals that the expected (80 kDa) and the lower (70 kDa) bands are detected in normal liver, whereas at day 10 after PH, the expected band is solely recognized, showing a different processing pattern of DGKα in liver regeneration. These results suggest that DGKζ and DGKα are involved, respectively, in the nucleus and the cytoplasm of hepatocytes in regenerating liver.