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.

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.

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.

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 oxidative DNA damage correlates with iron overload in chronic hepatitis C patients.

Hepatic oxidative stress occurs in chronic hepatitis C (CH-C), but little is known about its producing mechanisms and precise role in the pathogenesis of the disease. To determine the relevance of hepatic oxidatively generated DNA damage in CH-C, 8-hydroxy-2'-deoxyguanosine (8-OHdG) adducts were quantified in liver biopsy specimens by immunohistochemical staining, and its relationship with clinical, biochemical, and histological parameters, and treatment response was assessed in 40 CH-C patients. Hepatic 8-OHdG counts were significantly correlated with serum transaminase levels (r=0.560, p=0.0005) and histological grading activity (p=0.0013). Remarkably, 8-OHdG levels were also significantly related to body and hepatic iron storage markers (vs serum ferritin, r=0.565, p=0.0004; vs hepatic total iron score, r=0.403, p=0.0119; vs hepatic hepcidin messenger RNA, r=0.516, p=0.0013). Baseline hepatic oxidative stress was more prominent in nonsustained virological responder (non-SVR) than in SVR to interferon (IFN)/ribavirin treatment (50.8 vs 32.7 cells/10(5) microm2, p=0.0086). After phlebotomy, hepatic 8-OHdG levels were significantly reduced from 53.4 to 21.1 cells/10(5) microm2 (p=0.0125) with concomitant reductions of serum transaminase and iron-related markers in CH-C patients. In conclusion, this study showed that hepatic oxidatively generated DNA damage frequently occurs and is strongly associated with increased iron deposition and hepatic inflammation in CH-C patients, suggesting that iron overload is an important mediator of hepatic oxidative stress and disease progression in chronic HCV infection.

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.

Features of early gastric cancer and gastric adenoma by enhanced-magnification endoscopy.

BACKGROUND: Changes to the mucosal surface of early gastric carcinomas and gastric adenomas as viewed by enhanced-magnification endoscopy with acetic acid have not been investigated thoroughly. Using this technology, we investigated the appearance of the gastric surface patterns of neoplastic and surrounding nonneoplastic mucosa. METHODS: Forty-seven consecutive patients with early gastric carcinomas or gastric adenomas underwent enhanced-magnification endoscopy following 1.5% acetic acid instillation. All biopsy specimens were taken from the area at which the enhanced-magnified endoscopic image was obtained. RESULTS: Surface patterns of gastric tumors and the surrounding mucosa were classified into five types: type I, small round pits of uniform size and shape; type II, slit-like pits; type III, gyrus and villous patterns; type IV, irregular arrangements and sizes of pattern types I, II and III; type V, destructive patterns of types I, II and III. The predominant pattern of the surrounding mucosa was type III, and most type III mucosa had characteristics of intestinal metaplasia. Although all elevated adenomas showed type II or type III surface patterns, both depressed adenomas showed type IV. Elevated carcinomas showed type III (42.9%) or type IV (57.1%) surface patterns, while depressed carcinomas showed type IV (70%) or type V (30%). Although differentiated tubular adenocarcinomas showed type III (10.3%), type IV (86.2%), or type V (3.5%) surface patterns, all of the signet-ring cell carcinomas and poorly differentiated tubular adenocarcinomas showed type V. CONCLUSIONS: Enhanced-magnification endoscopy may be useful for identifying gastric tumors and determining the extent of horizontal spread, especially in tumors of the depressed type.

Increased oxidative stress is associated with decreased circulating levels of adiponectin in Japanese metabolically obese, normal-weight men with normal glucose tolerance.

To investigate the relationship between oxidative stress and circulating levels of adiponectin in Japanese metabolically obese, normal-weight [MONW; BMI<25 and visceral fat area; VFA > or =100 cm2 by abdominal computed tomography (CT) scanning] men with normal glucose tolerance (NGT), we measured the plasma levels of free 8-epi-prostaglandin F2alpha (8-epi-PGF2alpha) and adiponectin in 28 MONW and 23 normal men. The plasma levels of free 8-epi-PGF2alpha were measured using a commercially available enzyme immunoassay (EIA) kit (Cayman Chemical, Ann Arbor, MI). The plasma levels of adiponectin were measured using a radioimmunoassay kit (LINCO Research, St. Charles, MO). Plasma levels of 8-epi-PGF2alpha in MONW subjects (30.4+/-4.0 pg/ml; P<0.01) were significantly increased compared to controls (8.1+/-1.3 pg/ml). The plasma levels of adiponectin were significantly decreased in MONW subjects (8.6+/-0.9 microg/ml; P<0.01) as compared to normal subjects (11.6+/-0.6 microg/ml). The plasma levels of 8-epi-PGF2alpha and adiponectin were significantly correlated in MONW (r=-0.617, P<0.01) and in all (MONW+normal) (r=-0.620, P<0.01) subjects. The plasma levels of 8-epi-PGF2alpha and adiponectin were significantly correlated after adjustment for VFA in MONW subjects (F=11.042, P<0.01). The present study showed that systemic increase in oxidative stress correlates with decreased circulating levels of adiponectin in Japanese MONW men with NGT. Although correlation does not prove causation, this observation suggests that increased oxidative stress may decrease the production of adiponectin in Japanese MONW men with NGT.

Different metabolic correlations of thrombin-activatable fibrinolysis inhibitor and plasminogen activator inhibitor-1 in non-obese type 2 diabetic patients.

We investigated the plasma levels of thrombin-activatable fibrinolysis inhibitor (TAFI), plasminogen activator inhibitor-1 (PAI-1) and their relation with clinical and metabolic parameters in non-obese type 2 diabetic patients. The plasma levels of TAFI and PAI-1 were evaluated in 47 non-obese type 2 diabetic patients and 31 normal subjects. The intra-abdominal visceral and subcutaneous fat areas were measured by computed tomography (CT). The degree of insulin resistance was evaluated by the euglycemic-hyperinsulinemic clamp technique using artificial pancreas. The plasma levels of TAFI (169.0+/-108.8% versus 103.7+/-52.3%; p<0.001, mean+/-S.D.) and PAI-1 (82.7+/-54.5ng/ml versus 52.9+/-51.7ng/ml; p<0.05) were significantly higher in non-obese type 2 diabetic patients than in normal subjects. Univariate analysis showed that the plasma TAFI levels are significantly and inversely correlated with the glucose infusion rate (GIR) (r=-0.42, p<0.005) in all diabetic patients. Moreover, the plasma levels of TAFI were significantly correlated with fasting plasma glucose levels (r=0.47, p<0.001) and HbA(1c) (r=0.38, p<0.005) in all subjects. The plasma levels of PAI-1 were significantly and proportionally correlated with the visceral fat area (r=0.42, p<0.005) and body mass index (r=0.33, p<0.05). There was no significant correlation between plasma levels of TAFI and PAI-1 (r=0.04). These results show that the plasma levels of TAFI and PAI-1 differently correlate with insulin resistance and visceral fat accumulation, suggesting that different factors are implicated in the plasma elevation of TAFI and PAI-1 in non-obese type 2 diabetes mellitus.

Morphological identification of hepatitis C virus E1 and E2 envelope glycoproteins on the virion surface using immunogold electron microscopy.

It is known that hepatitis C virus (HCV) particles are spherical, 55-65 nm particles with fine surface projections of about 6 nm in length and with a 30-35 nm inner core. We have reported that free HCV particles labeled with gold particles specific to the HCV E1 glycoprotein are located in 1.14-1.16 g/ml fractions from plasma samples with high HCV RNA titers after sucrose density gradient centrifugation. However, the morphology of the HCV E2 glycoprotein on the virion has not yet been elucidated. To visualize HCV E2 localization on the virion, we used the same plasma samples where HCV particles were clearly shown. An indirect immunogold electron microscopic study was carried out using monoclonal and polyclonal anti-HCV E2 antibodies. HCV-like particles specifically reacted with the anti-HCV E2 antibodies. Moreover, to evaluate the localization of the HCV E1 and E2 glycoproteins on the virion surface, an immunogold electron microscopic study using double labeling with anti-HCV E1 antibodies and anti-HCV E2 antibodies was also performed. These particles also specifically reacted with both anti-E1 and E2 antibodies. This is the first report showing the presence of both HCV E1 and E2 glycoproteins on HCV virion surface in human plasma samples.

Lactoferrin inhibits lipid peroxidation in patients with chronic hepatitis C.

Lactoferrin is a milk protein with inhibitory effect on lipid peroxidation induced by oxidative stress. Oxidative stress plays an important role in the pathogenesis of chronic hepatitis C (CHC). The aim of this study was to evaluate the effect of bovine lactoferrin (bLF) monotherapy on lipid peroxidation, hepatic inflammation and iron metabolism in patients with CHC. Ninety Japanese patients with CHC were randomly assigned to two groups: bLF group (n=47) treated with bLF at a dose of 3.6g/day and a control group (n=43) that remained untreated. Plasma 8-isoprostane levels and clinical laboratory data including iron metabolism parameters were measured. Plasma 8-isoprostatne level was significantly decreased from 8.6+/-3.7 to 6.9+/-2.1pg/ml in the bLF group (P<0.05). Plasma levels of 8-isoprostane did not significantly change in the control group. The decline in plasma 8-isoprostane levels was positively correlated with improvement in the level of ALT in the bLF group. No significant change in serum HCV RNA levels or iron metabolism markers was found after bLF treatment. Therapy with bLF was associated with improvement in lipid peroxidation and ALT levels in CHC. Administration of bLF is a promising therapeutic approach for suppressing oxidative stress in non-responders to antiviral therapy.

Polymorphism of UDP-glucuronosyltransferase 1A7 gene: a possible new risk factor for lung cancer.

UDP-glucuronosyltransferase (UGT) 1A7 detoxifies hydroxylated benzo-(alpha)-pyrenes and 2-hydroxyamino-1-methyl-6-phenylimidazo (4,5-beta) pyridine. The purpose of this study was to evaluate whether UGT1A7 polymorphisms are risk factors for lung cancer. A total of 113 Japanese patients with lung cancer and 178 healthy individuals were enrolled in this study. Genomic DNA was isolated from leukocytes. Exon 1 of UGT1A7 was sequenced. Homozygous UGT1A7*3/3 was observed in 17 (15%) of patients with lung cancer, and this incidence was significantly increased compared with the control group (4.5%, P=0.0036). Multivariate logistic regression analysis demonstrated a significant association of lung cancer with Brinkmann index (odds ratio=4.577, P=0.0004) and homozygous UGT1A7*3 (odds ratio=4.020, P=0.0037). The presence of UGT1A7 polymorphisms was associated with lung cancer. Homozygous UGT1A7*3 is a possible risk factor for lung cancer, at least in the Japanese population. Thus, determination of UGT1A7 polymorphisms may provide an important clue to preventive measures against lung cancer.

Decreased circulating levels of active ghrelin are associated with increased oxidative stress in obese subjects.

OBJECTIVE: To investigate the relationship between active ghrelin and oxidative stress in obese subjects. DESIGN: We measured the plasma levels of free 8-epi-prostaglandin F(2alpha) (8-epi-PGF(2alpha), a reliable and systemic marker of oxidative stress) and the active form of ghrelin in 17 obese and 17 normal subjects. The biologically active forms of ghrelin were measured using a commercially available radio-immunoassay kit and free 8-epi-PGF(2alpha) was measured using an enzyme immunoassay kit. RESULTS: The circulating level of active ghrelin was significantly decreased (20.4 +/- 2.6 vs 40.9 +/- 3.9 fmol/ml, P < 0.01) while that of 8-epi-PGF(2alpha) was significantly increased (61.5 +/- 9.6 vs 17.3 +/- 3.4 pg/ml, P < 0.01) in obese subjects compared with normal subjects. The plasma levels of active ghrelin and 8-epi-PGF(2alpha) were significantly correlated in obese (r = -0.507, P < 0.05) and in all (r = -0.577, P < 0.01) subjects. Multivariate analysis showed that the plasma levels of active ghrelin and 8-epi-PGF(2alpha) were significantly and independently correlated in all subjects (F = 7.888, P < 0.01). CONCLUSIONS: There is an inverse correlation between circulating levels of active ghrelin and oxidative stress in obesity. Low circulating levels of active ghrelin may enhance oxidative stress and the process of atherosclerosis in obese subjects.

Transfecting the multidrug resistance protein 2 gene improves transcellular organic anion transport.

Eisai hyperbilirubinuria rats (EHBRs) lack functionally active multidrug resistance protein 2 (Mrp2), which causes impaired biliary excretion of numerous organic anions. We previously reported that Mrp3 expression is enhanced while organic anion transporting polypeptide 1 (Oatp1) or Oatp2 expression is reduced in the liver of EHBRs. Mrp3 mediates basolateral efflux of organic anions but not canalicular export. In this study, we transfected the human MRP2 gene into the liver of EHBRs and evaluated whether its transfection improves transcellular transport of organic anions in hepatocytes of EHBRs. The protein expression vector (pDEST26) including the full-length human MRP2 cDNA was developed. This vector was mixed with the hemagglutinating virus of Japan (HJV) envelope protein and transfected into the liver of EHBRs via the portal vein. Expression of Mrp3, Oatp1 and Oatp2 was evaluated by reverse transcription-polymerase chain reaction and Western blot analysis. mRNA and protein expression of MRP2 were detected in hepatocytes from transfected EHBRs. The serum-conjugated bilirubin level in EHBRs decreased to a normal level (35.7 to 6.4 micromol/l) with the expression of human MRP2. The change in expression of Mrp3, Oatp1 and Oatp2 in the liver of EHBRs was normalized by transfecting MRP2. Transfection of human MRP2 was performed using the HJV envelope protein. Transfection of MRP2 is useful for improving the transcellular transport of organic anions in the livers of EHBRs.