The Impact of a Heterozygous SLCO1B3 Null Variant on the Indocyanine Green Retention Test.

Indocyanine green (ICG) retention test is widely used for preoperative evaluation of liver function. OATP1B3 (gene symbol: SLCO1B3) is the major transporter for hepatic ICG uptake. We previously demonstrated that the individuals with a homozygous SLCO1B3 null allele revealed markedly impaired ICG clearance. However, the effect of heterozygosity of this variant on ICG clearance remains unknown. We compared the results of ICG retention rate at 15 min (ICG-R15) and hepatic OATP1B3 expression among individuals whose SLCO1B3 genotype was determined. Although OATP1B3 expression was significantly lower in the heterozygosity than the wild-type, the ICG-R15 results were comparable; 8.4 ± 3.4 (mean ± SD) % in the heterozygosity and 8.7 ± 6.0% in the wild-type. A homozygous individual revealed markedly high ICG-R15 (79.8%) and lacked OATP1B3 expression. In conclusion, the individuals with a heterozygous SLCO1B3 null variant had similar ICG clearance capacity to those with the wild-type despite decreased hepatic OATP1B3 expression.

Recessive inheritance of population-specific intronic LINE-1 insertion causes a rotor syndrome phenotype.

Sequences of long-interspersed elements (LINE-1, L1) make up ∼17% of the human genome. De novo insertions of retrotransposition-active L1s can result in genetic diseases. It has been recently shown that the homozygous inactivation of two adjacent genes SLCO1B1 and SLCO1B3 encoding organic anion transporting polypeptides OATP1B1 and OATP1B3 causes a benign recessive disease presenting with conjugated hyperbilirubinemia, Rotor syndrome. Here, we examined SLCO1B1 and SLCO1B3 genes in six Japanese diagnosed with Rotor syndrome on the basis of laboratory data and laparoscopy. All six Japanese patients were homozygous for the c.1738C>T nonsense mutation in SLCO1B1 and homozygous for the insertion of a ∼6.1-kbp L1 retrotransposon in intron 5 of SLCO1B3, which altogether make up a Japanese-specific haplotype. RNA analysis revealed that the L1 insertion induced deleterious splicing resulting in SLCO1B3 transcripts lacking exon 5 or exons 5-7 and containing premature stop codons. The expression of OATP1B1 and OATP1B3 proteins was not detected in liver tissues. This is the first documented case of a population-specific polymorphic intronic L1 transposon insertion contributing to molecular etiology of recessive genetic disease. Since L1 activity in human genomes is currently seen as a major source of individual genetic variation, further investigations are warranted to determine whether this phenomenon results in other autosomal-recessive diseases.

Regional reduction in gray and white matter volume in brains of cirrhotic patients: voxel-based analysis of MRI.

Chronic hepatic encephalopathy is a characteristically reversible neuropsychiatric disorder that occurs mainly in patients with liver cirrhosis. The brain regions critically involved in the pathophysiology of cirrhosis are not clear. Magnetic resonance imaging (MRI) with voxel-based morphometry (VBM) is a valuable tool for evaluating structural brain changes in many neurodegenerative diseases. We performed an MRI scan on 18 patients with liver cirrhosis and 16 age-matched healthy controls. We evaluated brain regional structural changes, regional differences and the relationship of these changes with the blood levels of ammonia and the results of neuropsychological tests in patients with cirrhosis. The VBM showed reduction in the volume of gray matter in the cerebellum and occipital lobe and in the volume of white matter in the cingulate, parietal, temporal, occipital lobe and precentral area in cirrhotic patients compared with controls. There were significant correlations between the volume of these regions with the plasma levels of ammonia and the results of neuropsychological tests. Voxel-based analysis of MRI revealed evidence for structural abnormalities of brain in patients with cirrhosis. Abnormal function in the above regions may account for the ammonia-mediated changes and neuropsychological deficits in hepatic encephalopathy.

Positron emission tomography image on evaluating intraperitoneal dissemination of malignant gastrointestinal stromal tumor.

Herein is a report of a patient with gastrointestinal stromal tumor (GIST) possibly arising from greater omentum accompanying diffuse peritoneal disseminatation. Positron emission tomography (PET) with 18F-fluorodeoxyglucose (18F-FDG) revealed that 18F-FDG uptake was widely spreading in the abdomen. In this case, the PET image was more useful than computed tomography (CT) for understanding tumor distribution rather. PET provides important information on tumor distribution and has an impact on evaluating clinical stage in GIST patients.

Hepatic iron accumulation is associated with disease progression and resistance to interferon/ribavirin combination therapy in chronic hepatitis C.

BACKGROUND AND AIMS: Liver iron accumulation in patients with chronic hepatitis C (CHC) has received increasing attention in recent years. The aim of this study was to determine the prevalence and severity of liver iron deposition in CHC, to assess its relationship with clinical, biochemical and histological characteristics, and to study its influence on the response to interferon (IFN) plus ribavirin combination therapy. METHODS: We studied liver biopsy specimens from 103 hepatitis C virus (HCV) and 34 hepatitis B virus (HBV) infected patients and total iron score (TIS) was measured. Seventy patients infected with HCV genotype 1b were treated with IFN/ribavirin for 24 weeks. RESULTS: CHC patients had a significantly higher TIS than chronic hepatitis B (CHB) patients (7.03 +/- 5.34 vs 4.41 +/- 4.49, P = 0.0056). TIS was significantly correlated with alcohol intake (P = 0.0213, r = 0.290), transaminase level (P = 0.0126, r = 0.247), platelet count (P = 0.0002, r = -0.369), histological grading (P = 0.0121, r = 0.248) and staging (P = 0.0003, r = 0.356) in CHC patients. Pretreatment TIS was significantly higher in non-sustained virological responders (SVR) than in SVR to IFN/ribavirin treatment (TIS = 7.69 +/- 5.76 vs 4.39 +/- 3.27, P = 0.0310). Multiple regression analysis showed that TIS was the only independent variable associated with resistance to IFN/ribavirin (P = 0.0277). CONCLUSIONS: Liver iron deposition was common in CHC compared to CHB and was associated with liver disease progression. Increased hepatic iron stores in CHC were related to resistance to IFN/ribavirin treatment.

Effect of lactoferrin in patients with chronic hepatitis C: combination therapy with interferon and ribavirin.

OBJECTIVES: Lactoferrin has been reported to inhibit hepatitis C virus (HCV) infection in cultured human hepatocytes and HCV viremia in patients with chronic hepatitis C (CHC). The aim of this study was to evaluate the effect of combined triple therapy of lactoferrin, interferon and ribavirin in patients with CHC. METHODS: A total of 111 Japanese patients with CHC were randomly assigned to a lactoferrin group (n = 50) and a control group (n = 61). The lactoferrin group was treated with lactoferrin for 8 weeks and then with lactoferrin, interferon and ribavirin for 24 weeks; the control group was treated with interferon and ribavirin for 24 weeks. Serum anti-lactoferrin antibody, clinical and laboratory measurement were determined. RESULTS: The mean HCV RNA titer significantly decreased at the end of lactoferrin monotherapy. Sustained virological response to therapy was significantly higher (P < 0.05) in the lactoferrin responder group (55%) than in the control group (18%). CONCLUSIONS: The results show that the decrease in HCV RNA titer by lactoferrin monotherapy contributes to the effectiveness of the combined therapy of interferon and ribavirin in patients with CHC. Lactoferrin is a potential useful adjunct treatment for patients with CHC.

Inhibition of nuclear factor-kappaB in T cells suppresses lung fibrosis.

RATIONALE: Cytokines secreted by T cells play a pivotal role in the pathogenesis of lung injury and fibrosis, and the transcription factors nuclear factor (NF)-kappaB and activator protein (AP)-1 are involved in the expression of cytokines from T cells during lung injury. OBJECTIVES: We assessed the potential therapeutic effect of SP100030, a specific inhibitor of T-cell NF-kappaB and AP-1 in lung fibrosis. METHODS: The effect of SP100030 was evaluated using a mouse model of chronic lung fibrosis. MEASUREMENTS AND MAIN RESULTS: Mice treated with SP100030, as compared with untreated mice, had significantly less cachexia and less lung injury and had decreased levels of inflammatory cytokines and growth factors, decreased activation of coagulation activation, and decreased collagen deposition in the lung. The inhibitory activity of SP100030 was dose dependent and was effective in acute and chronic phases of lung fibrosis. SP100030 inhibited the activation of the protein kinase C-isoform in T-cell lines and suppressed NF-kappaB-driven cytokine expression in CD4(+) and CD8(+) T cells. CONCLUSIONS: These results suggest that the specific inhibition of NF-kappaB could be useful for the treatment of lung fibrosis.

Th1-type immune responses by Toll-like receptor 4 signaling are required for the development of myocarditis in mice with BCG-induced myocarditis.

The immunological aspects of autoimmune myocarditis are difficult to understand because of the existence of many infectious agents and animal models suggesting different mechanisms in autoimmune myocarditis. To overcome these difficulties, two strains of mice, C3H/HeN and C3H/HeJ, showing different immune responses to mycobacteria, were immunized with myosin mixed with BCG. The C3H/HeN mice with a wild-type Toll-like receptor 4 (TLR4) showed severe myocarditis, whereas the C3H/HeJ mice with nonfunctional mutated TLR4 did not. CD4(+) cells from both strains of mice exhibited appreciable proliferative responses following myosin stimulation; however, the cytokines from these cells differed between these two strains. The C3H/HeN mice showed T helper (Th)1-type cytokine responses, whereas the expressions of mRNA in C3H/HeJ mice were Th2-type cytokine. When both of these strains of immunized mice were inoculated with a plasmid encoding cDNA of interleukin (IL)-4 or agonistic IL-4, the development of myocarditis was inhibited in C3H/HeN mice. Moreover, C3H/HeJ mice, in which development of myocarditis was not induced by immunization of myosin mixed with BCG, showed myocarditis after injection of IL-4 antagonistic mutant DNA for the induction of Th1-type immune responses. The results suggested that the induction of autoimmune myocarditis by myosin is affected by Th1-type immune responses.

Ultrastructural demonstration of hepatitis B virus production in a mouse model produced by hydrodynamic transfection.

A mouse model of hepatitis B virus (HBV) infection produced by hydrodynamic injection of HBV DNA has been recently established. However, the ultrastructural demonstration of HBV particles in this mouse model has not as yet been reported. In our study, plasmid DNA containing wild-type HBV DNA was rapidly injected into 8-week-old female SCID mice via the tail vein. Serum levels of HBsAg were measured by ELISA kit. Intrahepatic HBV protein expression was detected by immunohistochemistry of HBcAg. Ultrastructural study of the serum samples was performed by transmission electron microscopy and immunogold electron microscopy. Serum HBsAg and intrahepatic HBcAg were detected in HBV DNA-injected mice for at least 14 days. Spherical and filamentous particles 22 nm in diameter and double-shelled Dane-like particles 42 nm in diameter were detected in the sera of mice. The ultrastructural features of these particles were identical to HBV particles observed in serum from chronic hepatitis B patients. These particles were confirmed to be HBV particles by immunogold electron microscopy. We conclude that our present HBV mouse model using hydrodynamic transfection of HBV DNA is appropriate for production of HBV virions including Dane particles. This mouse model may be useful for screening in vivo the efficacy of antiviral drugs.

Hepatic arterial infusion of 5-fluorouracil in combination with subcutaneous interferon-alpha for advanced hepatocellular carcinoma.

BACKGROUND/AIMS: Transcatheter arterial chemoinfusion (TACI) is the main therapeutic modality for advanced hepatocellular carcinoma (HCC) with portal thrombus. However, TACI is not sufficient to improve prognosis. In this study, we evaluated the response to hepatic arterial infusion of 5-fluorouracil (5-FU) in combination with subcutaneous interferon (IFN)-alpha in patients with advanced HCC. METHODOLOGY: Ten patients (men, 8; women, 2; mean age, 55-77) with advanced HCC were enrolled in this study. Hepatic arterial infusion of 5-FU (500 mg/24 hrs) was performed for 5 days on the first and second week. IFN-alpha (5 x 10(6) International Units) was subcutaneously administered three times a week for 4 weeks (1 therapeutic course). Response to therapy was evaluated by abdominal computed tomography at the end of two courses of therapy. RESULTS: Seven patients received more than two courses of therapy. One patient (14%) showed complete response (CR). Four patients had stable disease (SD) (57%) and the remaining 2 patients had progressive disease (PD) (29%). Tumor markers decreased in all patients except 1 with PD. The 6-month survival rate was 40%. Therapy was discontinued in 3 patients due to severe adverse effects; all of these patients were over 70 years old, and had moderate liver dysfunction (Child-Pugh score of Grade B) before initiation of therapy. CONCLUSIONS: The goal of the therapy with hepatic arterial infusion of 5-FU in combination with subcutaneous IFN-alpha was attained in only 14% among our advanced HCC patients. The tumor completely disappeared in 1 patient, suggesting that this therapeutic modality may be of potential benefit in advanced HCC patients. However, this therapy should be performed with caution in patients with poor hepatic function (grade B or C of Child-Pugh score) and in those more than 70 years old.

Hepcidin expression in the liver: relatively low level in patients with chronic hepatitis C.

Patients with chronic hepatitis C frequently have serum and hepatic iron overload, but the mechanism is unknown. Recently identified hepcidin, exclusively synthesized in the liver, is thought to be a key regulator for iron homeostasis and is induced by infection and inflammation. This study was conducted to determine the hepatic hepcidin expression levels in patients with various liver diseases. We investigated hepcidin mRNA levels of liver samples by real-time detection-polymerase chain reaction; 56 were hepatitis C virus (HCV) positive, 34 were hepatitis B virus (HBV) positive, and 42 were negative for HCV and HBV (3 cases of auto-immune hepatitis, 7 alcoholic liver disease, 13 primary biliary cirrhosis, 9 nonalcoholic fatty liver disease, and 10 normal liver). We analyzed the relation of hepcidin to clinical, hematological, histological, and etiological findings. Hepcidin expression levels were strongly correlated with serum ferritin (P < 0.0001) and the degree of iron deposit in liver tissues (P < 0.0001). Hepcidin was also correlated with hematological parameters (vs. hemoglobin, P = 0.0073; vs. serum iron, P = 0.0012; vs. transferrin saturation, P < 0.0001) and transaminase levels (P = 0.0013). The hepcidin-to-ferritin ratio was significantly lower in HCV(+) patients than in HBV(+) patients (P = 0.0129) or control subjects (P = 0.0080). In conclusion, hepcidin expression levels in chronic liver diseases were strongly correlated with either the serum ferritin concentration or degree of iron deposits in the liver. When adjusted by either serum ferritin values or hepatic iron scores, hepcidin indices were significantly lower in HCV(+) patients than in HBV(+) patients, suggesting that hepcidin may play a pivotal role in the pathogenesis of iron overload in patients with chronic hepatitis C.

Unidentified virus-like particles are detected in plasmas with elevated ALT levels: are they significant of etiological agent(s) of non-B, non-C hepatitis?

GB virus C (GBV-C) and hepatitis G virus (HGV) have been proposed as new viruses etiologically implicated in non-B, non-C hepatitis, but the morphology of these particular virus particles is still unknown, and most cases of non-A to E hepatitis do not relate to their infections. We tried to visualize virus-like particles (VLPs) in plasma samples from hepatitis B surface antigen- and antibody to hepatitis C virus (HCV)-negative blood donors with elevated alanine aminotransferase (ALT), and examined the association of the virus-like particles and the genomes of parenterally transmissible GBV-C/HGV. Twenty-three plasma samples, 13 with elevated ALT levels and 10 with normal ALT values, from blood donors without infections of hepatitis B virus (HBV) and HCV, were subjected to a 20%-60% sucrose density gradient centrifugation, and virus-like particles were observed by electron microscopy. GBV-C/HGV RNAs in the plasmas were tested. Virus-like particles were found in the fractions with densities of 1.15-1.16 g/ml from 12 of 13 (92.3%) plasmas with elevated ALT levels and 1 of 10 (10%) normal controls. The ultrastructural morphology of visualized VLPs was pleomorphic in size and appearance; the majority of the VLPs were 50- to 80-nm spherical particles with a 35- to 45-nm inner core and 9- to 12-nm-long surface spike-like projections. Rodlike VLPs 50-70 nm in diameter with a length of 110-160 nm were also observed in the same samples. The incidence of detection of the circulating VLPs was significantly (P < 0.001) related to elevated ALT levels, but GBV-C/HGV RNAs were detected in none of the plasmas containing the virus-like particles. Spherical VLPs are detected in HBV- and HCV-negative plasmas significantly correlated with the elevation of ALT, suggesting that they are implicated in non-B, non-C hepatitis.