Impaired mitochondrial ß-oxidation in patients with chronic hepatitis C: relation with viral load and insulin resistance.

BACKGROUND: Hepatic steatosis is often seen in patients with chronic hepatitis C (CH-C). It is still unclear whether these patients have an impaired mitochondrial ß-oxidation. In this study we assessed mitochondrial ß-oxidation in CH-C patients by investigating ketogenesis during fasting. METHODS: This study consisted of thirty patients with CH-C. Serum levels of insulin and hepatitis C virus (HCV) core protein were measured by chemiluminescence enzyme immunoassay. The subjects were then fasted, and venous blood samples were drawn 12 h and 15 h after the start of fasting. The levels of blood ketone bodies were measured by an enzymatic cycling method. The rate of change in total ketone body concentration was compared with that in eight healthy volunteers. RESULTS: The rate of change in total ketone body concentration between 12 h and 15 h after the start of fasting was significantly lower in CH-C patients than in healthy volunteers (129.9% (8.5-577.3%) vs. 321.6% (139.6-405.4%); P <0.01). The rate of change in total ketone body concentration in patients with a serum level of HCV core protein of 10000 fmol/L or higher was significantly lower than in patients with a level of less than 10000 fmol/L (54.8% (8.5-304.3%) vs. 153.6% (17.1-577.3%); P <0.05). The rate of change in total ketone body concentration in patients with a homeostasis model assessment of insulin resistance (HOMA-IR) of 2.5 or higher was significantly lower than in patients with a HOMA-IR of less than 2.5 (56.7% (8.5-186.7%) vs. 156.4% (33.3-577.3%); P <0.01). CONCLUSIONS: These results suggest that mitochondrial ß-oxidation is impaired, possibly due to HCV infection in patients with CH-C.

Structure-activity relationship studies of 5-benzylaminoimidazo[1,2-c]pyrimidine-8-carboxamide derivatives as potent, highly selective ZAP-70 kinase inhibitors.

Zeta-associated protein, 70 kDa (ZAP-70), a spleen tyrosine kinase (Syk) family kinase, is normally expressed on T cells and natural killer cells and plays a crucial role in activation of the T cell immunoresponse. Thus, selective ZAP-70 inhibitors might be useful not only for treating autoimmune diseases, but also for suppressing organ transplant rejection. In our recent study on the synthesis of Syk family kinase inhibitors, we discovered that novel imidazo[1,2-c]pyrimidine-8-carboxamide derivatives possessed potent ZAP-70 inhibitory activity with good selectivity for ZAP-70 over other kinases. In particular, compound 26 showed excellent ZAP-70 kinase inhibition and high selectivity for ZAP-70 over structurally related Syk. The discovery of a potent, highly selective ZAP-70 inhibitor would contribute a new therapeutic tool for autoimmune diseases and organ transplant medication.

[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.

Orally active factor Xa inhibitors: investigation of a novel series of 3-amidinophenylsulfonamide derivatives using an amidoxime prodrug strategy.

A series of novel and potent 3-amidinophenylsulfonamide derivatives of factor Xa inhibitors were designed and synthesized using an amidoxime prodrug strategy. We focused on systemic clearance of parent compounds in rats, and performed in vivo pharmacokinetic screening. Incorporation of a carboxymethoxy group markedly improved systemic clearance (compound 43), and the related amidoxime 44 showed sufficient prodrug conversion. Compound 45, the double prodrug of 43, exhibited practicable bioavailability after oral administration in rats. Among the various compounds under investigation, KFA-1982 was selected for clinical development.

Structure-activity relationship studies of imidazo[1,2-c]pyrimidine derivatives as potent and orally effective Syk family kinases inhibitors.

Spleen tyrosine kinase (Syk) and zeta-associated protein kinase of 70k Da (ZAP-70) are members of the Syk family and non-receptor-type protein tyrosine kinases, which play crucial roles in B- and T-cell activation. Therefore, a Syk family tyrosine kinases inhibitor would be a useful therapeutic agent for the treatment of various allergic disorders and autoimmune diseases. Previously, we reported that 1,2,4-triazolo[4,3-c]pyrimidine derivative 1 and 1,2,4-triazolo[1,5-c]pyrimidine derivative 2 showed strong inhibitory activities against Syk family kinases. These compounds also exhibited high-level suppression of IL-2 in cellular assays. However, their oral efficacies were poor in a mouse model of IL-2 production. To improve oral effectiveness, we investigated a new series of Syk family kinases inhibitors. We found that imidazo[1,2-c]pyrimidine derivatives potently inhibited the Syk family kinases. Among these agents, compound 9f not only showed strong inhibitory activities against Syk and ZAP-70 kinases in vitro, but its oral administration resulted in the in vivo suppression of both the passive cutaneous anaphylaxis reaction and Concanavalin A-induced IL-2 production in a mouse model.

A novel Syk family kinase inhibitor: design, synthesis, and structure-activity relationship of 1,2,4-triazolo[4,3-c]pyrimidine and 1,2,4-triazolo[1,5-c]pyrimidine derivatives.

Splenic tyrosine kinase (Syk) family kinases, which are members of the protein tyrosine kinase family, play crucial roles in immune responses, with Syk participating in B-cell activation and the zeta-associated protein 70 kDa (ZAP-70) kinase being involved in T-cell activation. Therefore, Syk family kinase inhibitors are candidate therapeutic agents for the treatment of various allergic disorders and autoimmune diseases. We designed 1,2,4-triazolo[4,3-c]pyrimidine and 1,2,4-triazolo[1,5-c]pyrimidine derivatives as Syk family kinase inhibitors, based on literature reports and structure-based drug design. These derivatives showed significant Syk inhibitory activities, with ZAP-70 inhibition. Representative compounds 10d and 11 not only exhibited strong inhibition of both Syk and ZAP-70 kinase but also suppressed IL-2 production by peripheral blood mononuclear cells and whole blood.

[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.

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.

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.

Highly selective detection of trace copper(II) using bathocuproinesulfonate by flow-injection electrospray ionization mass spectrometry.

Trace Cu(2+) was detected with high selectivity using specific complexation with bathocuproinesulfonate (BCS) through flow-injection electrospray ionization mass spectrometry (FI-ESI-MS), which separates Cu(2+) from coexisting metal ions by forming a Cu-BCS complex with a high mass number. Here, only [Cu(I)(BCS)2](3-) was obtained with a high ion count. Its calibration curve was linear from 1.0 × 10(-8) to 1.0 × 10(-5) M. This method was applied to determine the Cu-complexing capacities of humic acid solution and river water samples by adding traces of Cu(2+).

1. Fatty liver and non-alcoholic steatohepatitis.

Although steatohepatitis can be induced by an excessive intake of alcohol, it can also arise through various other causes, in which case it is known as non-alcoholic fatty liver disease (NAFLD). NAFLD is classified into two categories:simple fatty liver with a favorable clinical outcome, and non-alcoholic steatohepatitis (NASH), which is intractable and progressive. Recently in Japan, there has been an increase in the number of individuals at risk of lifestyle-related diseases, due to increased insulin resistance and visceral fat obesity. The metabolic syndrome (MS) is associated with several risk factors for atherosclerosis, including diabetes mellitus (DM), hypertension, and hyperlipidemia. Visceral fat obesity is the prime cause of NASH in the liver, and is therefore considered to be one of the phenotypic features of MS. Furthermore, most chronic liver diseases are associated with hepatitis C virus (HCV) infection. Fatty degeneration of hepatocytes is often observed in the liver of HCV-infected individuals, and results from viral suppression of mitochondrial beta-oxidation of fatty acid. The natural outcome of HCV infection is worse in patients with lifestyle-related high insulin resistance and visceral fat obesity. In this review, we describe the recent advances in research on progressive liver diseases that are the result of fat accumulation in the liver, with special reference to MS.

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.

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.