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Purpose . The aim of this study was to assess the repeatability of the SUV normalized by liver volume (SUVL) between two liver receptor SPECT/CT studies performed on different days in patients with ICG-R15 values within normal range. Methods . 935 patients who underwent liver receptor scintigraphy between January 2010 and August 2018 were included. Patients who underwent liver resection, hepatic arterial embolization or had ICG-R15 >10% between scans were excluded, and 38 patients were finally included in the analysis. The repeatability of SUVL between scans was assessed using the intraclass correlation coefficient (ICC) (1.1) between SUVLmax, SUVLpeak and SUVLmean at the first and second scan and the additive and proportional errors from the Bland-Altman analysis. Results . In ICC (1,1), SUVLmax, SUVLpeak and SUVLmean were all greater than 0.8, indicating almost perfect repeatability; neither additive nor proportional errors were observed in the Bland-Altman analysis. Conclusions . In patients with ICG-R15 values within the normal range, the SUV Liver (SUVL) between two liver receptor SPECT/CT studies performed on different days was repeatability over time. It was suggested that the SUVL of liver receptor scintigraphy could be an indicator that could be used for follow-up over time in the assessment of liver fibrosis., (© 2024 IOP Publishing Ltd.)

Recombinant thrombomodulin (rTM) is a novel anticoagulant and anti-inflammatory agent that inhibits secretion of high-mobility group box 1 (HMGB1) from liver. We evaluated the protective effects of rTM on hepatic ischemia-reperfusion injury in rats. Ischemia was induced by clamping the portal vein and hepatic artery of left lateral and median lobes of the liver. At 30 min before ischemia and at 6 h after reperfusion, 0.3 ml of saline (IR group) or 0.3 ml of saline containing 6 mg/kg body weight of rTM (IR-rTM group) was injected into the liver through inferior vena cava or caudate vein. Blood flow was restored at 60 min of ischemia. Blood was collected 30 min prior to induction of ischemia and before restoration of blood flow, and at 6, 12, and 24 h after reperfusion. All the animals were euthanized at 24 h after reperfusion and the livers were harvested and subjected to biochemical and pathological evaluations. Serum levels of ALT, AST, and HMGB1 were significantly lower after reperfusion in the IR-rTM group compared to IR group. Marked hepatic necrosis was present in the IR group, while necrosis was almost absent in IR-rTM group. Treatment with rTM significantly reduced the expression of TNF-α and formation of 4-hydroxynonenal in the IR-rTM group compared to IR group. The results of the present study indicate that rTM could be used as a potent therapeutic agent to prevent IR-induced hepatic injury and the related adverse events., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Purpose: Gadolinium-ethoxybenzyl-diethylenetriamine pentaacetic acid (Gd-EOB-DTPA)-enhanced magnetic resonance imaging (MRI) tends to show higher diagnostic accuracy than other modalities. There is a demand for computer-assisted detection (CAD) software for Gd-EOB-DTPA-enhanced MRI. Segmentation with high accuracy is important for CAD software. We propose a liver segmentation method for Gd-EOB-DTPA-enhanced MRI that is based on a four-dimensional (4D) fully convolutional residual network (FC-ResNet). The aims of this study are to determine the best combination of an input image and output image in our proposed method and to compare our proposed method with the previous rule-based segmentation method., Methods: We prepared a five-phase image set and a hepatobiliary phase image set as the input image sets to determine the best input image set. We also prepared a labeled liver image and labeled liver and labeled body trunk images as the output image sets to determine the best output image set. In addition, we optimized the hyperparameters of our proposed model. We used 30 cases to train our model, 10 cases to determine the hyperparameters of our model, and 20 cases to evaluate our model., Results: Our network with the five-phase image set and the output image set of labeled liver and labeled body trunk images showed the highest accuracy. Our proposed method showed higher accuracy than the previous rule-based segmentation method. The Dice coefficient of the liver region was 0.944 ± 0.018., Conclusion: Our proposed 4D FC-ResNet showed satisfactory performance for liver segmentation as preprocessing in CAD software.

Ingestion of high-fructose corn syrup (HFCS) is associated with the risk of both diabetes and obesity. Rare sugar syrup (RSS) has been developed by alkaline isomerization of HFCS and has anti-obesity and anti-diabetic effects. However, the influence of RSS on glucose metabolism has not been explored. We investigated whether long-term administration of RSS maintains glucose tolerance and whether the underlying mechanism involves hepatic glucokinase translocation. Wistar rats were administered water, RSS, or HFCS in drinking water for 10 weeks and then evaluated for glucose tolerance, insulin tolerance, liver glycogen content, and subcellular distribution of liver glucokinase. RSS significantly suppressed body weight gain and abdominal fat mass (p < 0.05). The glucose tolerance test revealed significantly higher blood glucose levels in the HFCS group compared to the water group, whereas the RSS group had significantly lower blood glucose levels from 90 to 180 min (p < 0.05). At 30, 60, and 90 min, the levels of insulin in the RSS group were significantly lower than those in the water group (p < 0.05). The amount of hepatic glycogen was more than 3 times higher in the RSS group than that in the other groups. After glucose loading, the nuclear export of glucokinase was significantly increased in the RSS group compared to the water group. These results imply that RSS maintains glucose tolerance and insulin sensitivity, at least partly, by enhancing nuclear export of hepatic glucokinase.

Ischemia-reperfusion (IR) injury is a major clinical problem and is associated with numerous adverse effects. GGsTop [2-amino-4{[3-(carboxymethyl)phenyl](methyl)phosphono}butanoic acid] is a highly specific and irreversible γ-glutamyl transpeptidase (γ-GT) inhibitor. We studied the protective effects of GGsTop on IR-induced hepatic injury in rats. Ischemia was induced by clamping the portal vein and hepatic artery of left lateral and median lobes of the liver. Before clamping, saline (IR group) or saline containing 1 mg/kg body wt of GGsTop (IR-GGsTop group) was injected into the liver through the inferior vena cava. At 90 min of ischemia, blood flow was restored. Blood was collected before induction of ischemia and prior to restoration of blood flow and at 12, 24, and 48 h after reperfusion. All the animals were euthanized at 48 h after reperfusion and the livers were harvested. Serum levels of alanine transaminase, aspartate transaminase, and γ-GT were significantly lower after reperfusion in the IR-GGsTop group compared with the IR group. Massive hepatic necrosis was present in the IR group, while only few necroses were present in the IR-GGsTop group. Treatment with GGsTop increased hepatic GSH content, which was significantly reduced in the IR group. Furthermore, GGsTop prevented increase of hepatic γ-GT, malondialdehyde, 4-hydroxynonenal, and TNF-α while all these molecules significantly increased in the IR group. In conclusion, treatment with GGsTop increased glutathione levels and prevented formation of free radicals in the hepatic tissue that led to decreased IR-induced liver injury. GGsTop could be used as a pharmacological agent to prevent IR-induced liver injury and the related adverse events., (Copyright © 2016 the American Physiological Society.)

During the arterial phase acquisition of Gd-EOB-DTPA examinations, use of a small volume of the Gd-EOB-DTPA may make it difficult the encoding center of the k-space, and produce blurring. The previous studies revealed the encoding technique of the k-space was one of the most important reasons. However, there is no report to discuss the reasons with quantitative evaluations. The purpose of this study was to quantitatively evaluate the characteristics of the artifacts using different k-space encoding techniques (centric-view ordering (CVO) and sequential-view ordering (SVO)) for liver dynamic MRI in computer simulation study. This simulation study consists of the following steps. First of all, the creation of a time intensity curve, and original simulation images at certain points among the one phase dynamic scanning. Secondly, creation-simulated MR echo data from the created original images using FFT, and encoding simulated k-space using the simulated MR echo data. Finally, a reconstruction of simulated dynamic MR images from the simulated k-space, and to evaluate each simulated MR images, we measured modulation transfer functions (MTFs) from the bar patterns of the reconstructed images. The results of the CVO simulation indicated that the bar patterns were blurring compared to the images encoded by the SVO. The results of the SVO simulation indicated that the bar patterns were not enhanced at late scan timings. In addition, the results of MTFs indicated that there was no edge enhancement at all scan timings and both encoding techniques. In conclusion, it is possible to quantitatively evaluate the characteristics of artifacts using MTF, which was measured by the bar patterns, in liver dynamic MRI.

The degree of liver fibrosis is strongly associated with the antiviral effect of interferon on chronic hepatitis C patients. In this study, the accuracy of acoustic radiation force impulse (ARFI) in assessing liver fibrosis and the association between liver stiffness using ARFI and antiviral effects were investigated. The 124 patients with chronic hepatitis C enrolled in this study included 94 with HCV genotype 1 and 40 (30%) with moderate fibrosis (METAVIR fibrosis score ≥ F2). Sixty-one patients received pegylated interferon (peg-IFN) plus ribavirin combination therapy and the treatment responses were assessed. The shear wave velocity (Vs value) by ARFI had a strong correlation with the histological fibrosis stage (P < 0.001). The AUROC of the Vs value, aspartate aminotransferase platelet ratio index and FIB4 for the diagnoses of moderate fibrosis (≥F2) were 0.890, 0.779, and 0.737, respectively. HCV genotype 1 patients with the TT allele of IL28B and with a low Vs value (<1.40 m/sec) who were treated with peg-IFN plus ribavirin therapy achieved a sustained virologic response at a rate of 79% (15/19), while all patients with the TG/GG allele of IL28B and a high Vs value (≥1.40 m/sec) experienced a non-virologic response (6/6). The Vs value measured by ARFI could not predict the treatment response for patients with HCV genotype 2. It is concluded that the combination of ARFI at cut off of 1.4 m/sec and IL28B may be useful for patients with chronic hepatitis C with genotype 1 treated with peg-IFN/ribavirin combination therapy., (© 2013 Wiley Periodicals, Inc.)

Background: Chronic ingestion of ethanol increases acetaldehyde and leads to the production of acetaldehyde-derived advanced glycation end-products (AA-AGE). We evaluated the toxicity of AA-AGE on hepatocytes and studied the role of AA-AGE in the pathogenesis of alcoholic liver disease (ALD)., Methods: Rat hepatocyte cultures were treated with N-ethyllysine (NEL) or AA-AGE and the cell viability was evaluated using MTT assay. Male Wistar rats were fed with liquid diet containing 5% ethanol for 8 weeks following normal diet for another 12 weeks. A group of animals was sacrificed at 4th, 6th, and 8th week and the remaining animals at 12th, 14th, 16th, 18th, and 20th week. The liver sections were stained for AA-AGE and 4-hydroxy-2-nonenal (4-HNE). Liver biopsy obtained from ALD patients was also stained for AA-AGE and 4-HNE., Results: Hepatocyte viability was significantly reduced in cultures treated with AA-AGE compared to NEL treated or control cultures. Severe fatty degeneration was observed during chronic administration of ethanol increasing from 4-8 weeks. The staining of AA-AGE and 4-HNE was correlated with the degree of ALD in both rat and human. In rats, hepatic fatty degeneration was completely disappeared and the staining for both AA-AGE and 4-HNE returned to normal at 12th week of abstinence. Staining for AA-AGE and 4-HNE was completely absent in normal human liver., Conclusions: The data demonstrated that AA-AGE is toxic to hepatocytes, but not NEL. Chronic ethanol ingestion produces AA-AGE and reactive oxygen species that contribute to the pathogenesis of ALD. Abstinence of alcohol results in complete disappearance of both AA-AGE and 4-HNE along with fatty degeneration suggesting that AA-AGE plays a significant role in the pathogenesis of ALD.

Unlabelled: The polymorphisms in the interleukin (IL)-28B (interferon-lambda [IFN]-λ3) gene are strongly associated with the efficacy of hepatitis C virus (HCV) clearance. Dendritic cells (DCs) sense HCV and produce IFNs, thereby playing some cooperative roles with HCV-infected hepatocytes in the induction of interferon-stimulated genes (ISGs). Blood dendritic cell antigen 3 (BDCA3)(+) DCs were discovered as a producer of IFN-λ upon Toll-like receptor 3 (TLR3) stimulation. We thus aimed to clarify the roles of BDCA3(+) DCs in anti-HCV innate immunity. Seventy healthy subjects and 20 patients with liver tumors were enrolled. BDCA3(+) DCs, in comparison with plasmacytoid DCs and myeloid DCs, were stimulated with TLR agonists, cell-cultured HCV (HCVcc), or Huh7.5.1 cells transfected with HCV/JFH-1. BDCA3(+) DCs were treated with anti-CD81 antibody, inhibitors of endosome acidification, TIR-domain-containing adapter-inducing interferon-β (TRIF)-specific inhibitor, or ultraviolet-irradiated HCVcc. The amounts of IL-29/IFN-λ1, IL-28A/IFN-λ2, and IL-28B were quantified by subtype-specific enzyme-linked immunosorbent assay (ELISA). The frequency of BDCA3(+) DCs in peripheral blood mononuclear cell (PBMC) was extremely low but higher in the liver. BDCA3(+) DCs recovered from PBMC or the liver released large amounts of IFN-λs, when stimulated with HCVcc or HCV-transfected Huh7.5.1. BDCA3(+) DCs were able to induce ISGs in the coexisting JFH-1-positive Huh7.5.1 cells. The treatments of BDCA3(+) DCs with anti-CD81 antibody, cloroquine, or bafilomycin A1 reduced HCVcc-induced IL-28B release, whereas BDCA3(+) DCs comparably produced IL-28B upon replication-defective HCVcc. The TRIF-specific inhibitor reduced IL-28B release from HCVcc-stimulated BDCA3(+) DCs. In response to HCVcc or JFH-1-Huh7.5.1, BDCA3(+) DCs in healthy subjects with IL-28B major (rs8099917, TT) released more IL-28B than those with IL-28B minor genotype (TG)., Conclusion: Human BDCA3(+) DCs, having a tendency to accumulate in the liver, recognize HCV in a CD81-, endosome-, and TRIF-dependent manner and produce substantial amounts of IL-28B/IFN-λ3, the ability of which is superior in subjects with IL-28B major genotype., (Copyright © 2012 American Association for the Study of Liver Diseases.)

The focus of this study was on the investigation of the accuracy of the fat fraction of the liver by use of single-breath-holding magnetic resonance spectroscopy (MRS) with T (2) correction. Single-voxel proton MRS was performed with several TE values, and the fat fraction was determined with and without T (2) correction. MRS was also performed with use of the point-resolved spectroscopy sequence in single breath holding. The T (2) values of both water and fat were determined separately at the same time, and the effect of T (2) on the fat fraction was corrected. In addition, MRS-based fat fractions were compared with the degree of hepatic steatosis (HS) by liver biopsy in human subjects. With T (2) correction, the MRI-derived fat fractions were in good agreement with the fat fractions in all phantoms, but the fat fractions were overestimated without T (2) correction. R (2) values were in good agreement with the preset iron concentrations in the phantoms. The MRI-derived fat fraction was well correlated with the degree of HS. Iron deposited in the liver affects the signal strength when proton MRS is used for detection of the fat signal in the liver. However, the fat signal can be evaluated more accurately when the T (2) correction is applied. Breath-holding MRS minimizes the respiratory motion, and it can be more accurate in the quantification of the hepatic fat fraction.

It has been established that the long-term infection of chronic hepatitis C leads to the increased risk of hepatic fibrosis and hepatocellular carcinoma. Currently, histological diagnosis by invasive and painful liver biopsy is the gold standard for evaluating the hepatic fibrosis stage. Because of a side effect or patient inability to cope with the pain, it is difficult to assess the fibrosis stage frequently using liver biopsy. Recently, instead of liver biopsy, many articles have been published showing the usefulness of ultrasound elastography to evaluate the stage of hepatic fibrosis. We also reported the usefulness of real-time tissue elastography (RTE) for liver fibrosis staging in 2007. However, in our previous report, fibrosis classification was performed manually and the number of patients involved was also small. In the current study, the fibrosis staging is performed automatically using software by characterizing the elastography images. We have also increased the number of patients from 64 to 310. Thus, the aim of this study is to increase objectivity by using a newly developed automatic analysis method. We obtain the Liver Fibrosis Index (LFI), which is calculated from image features of RTE images, using multiple regression analysis performed on clinical data of 310 cases as the training data set. The correlation coefficient obtained between the LFI and the stage of hepatic fibrosis was r = 0.68, and significant differences exist between all stages of fibrosis (p < 0.001). Our new method seems promising since it has the ability to diagnose fibrosis even in the presence of inflammation., (Copyright © 2013 S. Karger AG, Basel.)

A 30-year-old man was admitted to Osaka University Hospital for the treatment of gastric varices and assessment of indication for liver transplant. When he was 6 years old, liver dysfunction was pointed out and diagnosed as chronic inactive hepatitis by liver biopsy. At 13 years of age, the second liver biopsy proved congenital hepatic fibrosis (CHF). The third liver biopsy was performed when he was 30 years old, and the progression of hepatic fibrosis was confirmed. Besides CHF, we recognized oligophrenia, cerebellar ataxia, hypoplasia of cerebellar vermis and coloboma, leading to the diagnosis of COACH syndrome. COACH syndrome is quite rare, and our case is especially valuable because he was diagnosed as an adult case and the progression of hepatic fibrosis could be followed through several liver biopsies. We should be aware of COACH syndrome in mind when we encounter CHF patients.

Background: Invariant natural killer T (iNKT) cells have been suggested to play critical roles in a wide range of immune responses by acting in a proinflammatory or anti-inflammatory manner. Nonalcoholic steatohepatitis (NASH) is a chronic liver disease progressing to advanced cirrhosis and hepatocellular carcinoma. Despite the abundance of iNKT cells in the liver, their role in the pathogenesis of NASH remains obscure. Here, we investigated their role in the development of diet-induced steatosis/steatohepatitis., Methods: We used BALB/c wild-type mice and Jα18-deficient (KO) mice lacking iNKT cells fed either a normal diet or a high-fat diet (HFD). The liver and blood were collected from these mice to examine liver inflammation, steatosis, and fibrosis at the indicated time points., Results: KO mice fed the HFD, compared with control mice fed the HFD, exhibited a clearly higher serum alanine aminotransferase level and a greater number of hepatic inflammatory foci, although there was no significant difference in hepatic lipid retention between these groups of mice. The HFD enhanced hepatic messenger RNA expression of inflammatory cytokines and chemokines in KO but not in control mice. The HFD also increased the proportion of hepatic CD4 T cells and CD8 T cells that composed hepatic inflammatory foci in KO mice, but not in the controls. Prolonged feeding with the HFD augmented liver fibrosis in KO but not in control mice., Conclusions: These findings indicate that iNKT cells play a protective role against liver inflammation progressing to fibrosis, but not against steatosis, enhanced by dietary excess fat, suggesting a key role of these cells in NASH pathogenesis.

Gd-EOB-DTPA is a new liver specific MRI contrast media. In the hepatobiliary phase, contrast media is trapped in normal liver tissue, a normal liver shows high intensity, tumor/liver contrast becomes high, and diagnostic ability improves. In order to indicate the degree of uptake of the contrast media, the enhancement ratio (ER) is calculated. The ER is obtained by calculating (signal intensity (SI) after injection-SI before injection) / SI before injection. However, because there is no linearity between contrast media concentration and SI, ER is not correctly estimated by this method. We discuss a method of measuring ER based on SI and T(1) values using the phantom. We used a column phantom, with an internal diameter of 3 cm, that was filled with Gd-EOB-DTPA diluted solution. Moreover, measurement of the T(1) value by the IR method was also performed. The ER measuring method of this technique consists of the following three components: 1) Measurement of ER based on differences in 1/T(1) values using the variable flip angle (FA) method, 2) Measurement of differences in SI, and 3) Measurement of differences in 1/T(1) values using the IR method. ER values calculated by these three methods were compared. In measurement made using the variable FA method and the IR method, linearity was found between contrast media concentration and ER. On the other hand, linearity was not found between contrast media concentration and SI. For calculation of ER using Gd-EOB-DTPA, a more correct ER is obtained by measuring the T(1) value using the variable FA method.

Background and Aim: Adiponectin is an anti-inflammatory and insulin-sensitizing adipocytokine, and its serum concentrations are reduced in obesity with visceral fat accumulation. Visceral fat accumulation is an independent determinant of elevated serum liver enzymes. Hypoadiponectinemia plays important roles in the clinical progression of nonalcoholic steatohepatitis. The aim of this study was to evaluate the relation between visceral fat area (VFA), serum adiponectin concentration, and biochemical liver tests, such as aspartate aminotransferase, alanine aminotransferase (ALT), and gamma-glutamyltransferase (GGT) in normal subjects., Methods: The study group comprised 3827 Japanese subjects [mean age+/-SD; 47.6+/-10.7 y: 2854 males (48.4+/-10.7 y), 973 females (45.3+/-10.1 y)], who underwent annual health checkup in 2004. In addition to parameters measured in the annual health checkup, VFA and serum adiponectin concentration were measured by the bioelectrical impedance analysis method and a latex particle-enhanced turbidimetric assay system, respectively., Results: Pearson's correlation analysis showed a significant correlation between VFA and the levels of the above 3 liver enzymes in both sexes, and a significant negative correlation between adiponectin and all biochemical liver tests in men and with ALT and GGT in women. Stepwise multiple regression analysis showed that VFA was a significant determinant of serum liver tests in both sexes. Moreover, serum adiponectin concentration significantly and negatively influenced male ALT and GGT and female GGT., Conclusions: Both visceral obesity and hypoadiponectinemia are significant determinants of subtle and asymptomatic hepatic dysfunction in normal Japanese subjects.

Unlabelled: Anti-apoptotic members of the Bcl-2 family, including Bcl-2, Bcl-xL, Mcl-1, Bcl-w and Bfl-1, inhibit the mitochondrial pathway of apoptosis. Bcl-xL and Mcl-1 are constitutively expressed in the liver. Although previous research established Bcl-xL as a critical apoptosis antagonist in differentiated hepatocytes, the significance of Mcl-1 in the liver, especially in conjunction with Bcl-xL, has not been clear. To examine this question, we generated hepatocyte-specific Mcl-1-deficient mice by crossing mcl-1(flox/flox) mice and AlbCre mice and further crossed them with bcl-x(flox/flox) mice, giving Mcl-1/Bcl-xL-deficient mice. The mcl-1(flox/flox) AlbCre mice showed spontaneous apoptosis of hepatocytes after birth, as evidenced by elevated levels of serum alanine aminotransferase (ALT) and caspase-3/7 activity and an increased number of terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate nick-end labeling (TUNEL)-positive cells in the liver; these phenotypes were very close to those previously found in hepatocyte-specific Bcl-xL-deficient mice. Although mcl-1(flox/+) AlbCre mice did not display apoptosis, their susceptibility to Fas-mediated liver injury significantly increased. Further crossing of Mcl-1 mice with Bcl-xL mice showed that bcl-x(flox/+) mcl-1(flox/+) AlbCre mice also showed spontaneous hepatocyte apoptosis similar to Bcl-xL-deficient or Mcl-1-deficient mice. In contrast, bcl-x(flox/flox) mcl-1(flox/+) AlbCre, bcl-x(flox/+) mcl-1(flox/flox) AlbCre, and bcl-x(flox/flox) mcl-1(flox/flox) AlbCre mice displayed a decreased number of hepatocytes and a reduced volume of the liver on day 18.5 of embryogenesis and rapidly died within 1 day after birth, developing hepatic failure evidenced by increased levels of blood ammonia and bilirubin., Conclusion: Mcl-1 is critical for blocking apoptosis in adult liver and, in the absence of Bcl-xL, is essential for normal liver development. Mcl-1 and Bcl-xL are two major anti-apoptotic Bcl-2 family proteins expressed in the liver and cooperatively control hepatic integrity during liver development and in adult liver homeostasis in a gene dose-dependent manner.

Background/aims: Alpha-galactosylceramide (alpha-GalCer) presented by dendritic cells (DCs) activates NKT cells that in turn drive DC maturation. However, the potential of generating acquired immunity of liver DCs in alpha-GalCer treatment remains unclear., Methods: We examined the activation of acquired immunity in the alpha-GalCer treatment against liver or spleen tumor and the ability of liver and spleen DCs in the generation of acquired immunity., Results: Administration of alpha-GalCer resulted in generation of p53 peptide-specific cytotoxic T lymphocytes (CTLs) in mice bearing liver CMS4 tumor, aberrantly expressing p53, but not in mice bearing spleen CMS4 tumor. The growth of rechallenged CMS4 subcutaneous tumor was inhibited in alpha-GalCer-treated mice against liver CMS4 tumor, but not in alpha-GalCer-treated mice against CMS4 spleen tumor. The antigen presenting related functions of liver DCs were significantly higher than those of spleen DCs in alpha-GalCer-treated mice. Vaccination of normal mice with p53 peptide pulsed liver DCs isolated from alpha-GalCer treated mice resulted in generation of p53 peptide-specific CTLs, but that with p53 peptide pulsed spleen DCs did not., Conclusions: These results demonstrated that alpha-GalCer treatment induced unique immunologic activation of liver DCs in comparison with spleen DCs, which might be favorable to generate liver acquired immunity.

Adipose tissue-derived stromal cells (ADSC) potentially differentiate into various cell types similar to bone marrow-derived mesenchymal stromal cells (BMSC). Unlike BMSC, ADSC can be harvested easily and repeatedly. However, the advantages of ADSC for cell transplantation in liver disease remain unclear. To investigate this, we developed a novel culture system for ADSC, as well as effective methods for transplantation of ADSC into mice liver. ADSC were isolated from subcutaneous adipose tissues of male C57BL6/J mice and cultured on plastic dishes with or without basic fibroblast growth factor (bFGF). In the in vivo study, ADSC isolated from green fluorescent protein-transgenic mice were transplanted into carbon tetrachloride-injured C57BL6/J mice liver. bFGF-treated ADSC expressed several liver-specific marker genes and demonstrated liver-related functions such as albumin secretion, glycogen synthesis, urea production, and low-density lipoprotein uptake. Importantly, pretreatment of ADSC with bFGF for 1 wk enhanced the repopulation rate of ADSC in mice liver, attenuated liver fibrosis, and restored normal serum alanine aminotransferase and albumin levels. The results indicate that basic FGF facilitates transdifferentiation of ADSC into hepatic lineage cells in vitro and that transplantation of bFGF-pretreated ADSC reduced hepatic fibrosis in mice. ADSC are a potentially valuable source of cells for transplantation therapy.

We previously demonstrated that adiponectin has anti-fibrogenic and anti-inflammatory effects in the liver of mouse models of various liver diseases. However, its role in liver regeneration remains unclear. The aim of this study was to determine the role of adiponectin in liver regeneration. We assessed liver regeneration after partial hepatectomy in wild-type (WT) and adiponectin knockout (KO) mice. We analyzed DNA replication and various signaling pathways involved in cell proliferation and metabolism. Adiponectin KO mice exhibited delayed DNA replication and increased lipid accumulation in the regenerating liver. The expression levels of peroxisome proliferator-activated receptor (PPAR) alpha and carnitine palmitoyltransferase-1 (CPT-1), a key enzyme in mitochondrial fatty acid oxidation, were decreased in adiponectin KO mice, suggesting possible contribution of altered fat metabolism to these phenomena. Collectively, the present results highlight a new role for adiponectin in the process of liver regeneration.

Unlabelled: Sepsis is an infection-induced syndrome with systemic inflammatory response leading to multiorgan failure and occasionally death. During this process, signal transducer and activator of transcription 3 (STAT3) is activated in the liver, but the significance of this molecule has not been established. We generated hepatocyte-specific STAT3-deficient mice (L-STAT3 KO) and examined the susceptibility of these mice to cecal ligation and puncture-induced peritonitis, a well-established septic model. L-STAT3 KO mice showed significantly higher mortality and produced lesser amounts of various acute phase proteins than control littermates. Although blood bacterial infection did not differ between L-STAT3 KO mice and control mice, the former showed deterioration of the systemic inflammatory response as evidenced by a significant increase in various cytokines such as tumor necrosis factor alpha, IFN-gamma, IL-6, IL-10, monocyte chemoattractant protein 1, and macrophage inflammatory protein 1beta. A similar hyperinflammatory response was observed in another septic model caused by lipopolysaccharide (LPS) injection. In vitro analysis revealed that soluble substances derived from hepatocytes and dependent on STAT3 were critical for suppression of cytokine production from LPS-stimulated macrophage and splenocytes., Conclusion: STAT3 activation in hepatocytes can attenuate a systemic hyperinflammatory response and lethality in sepsis, in part by suppressing immune cell overactivation, implying a critical role of hepatocyte STAT3 signaling in maintaining host homeostasis.

Aim: To explore whether a co-culture of cynomolgus monkey embryonic stem (cES) cells with embryonic liver cells could promote their differentiation into hepatocytes., Methods: Mouse fetal liver-derived cells (MFLCs) were prepared as adherent cells from mouse embryos on embryonic d (ED) 14, after which undifferentiated cES cells were co-cultured with MFLCs. The induction of cES cells along a hepatic lineage was examined in MFLC-assisted differentiation, spontaneous differentiation, and growth factors (GF) and chemicals-induced differentiations (GF-induced differentiation) using retinoic acid, leukemia inhibitory factor (LIF), FGF2, FGF4, hepatocyte growth factor (HGF), oncostatin M (OSM), and dexamethasone., Results: The mRNA expression of alpha-fetoprotein, albumin (ALB), alpha-1-antitrypsin, and hepatocyte nuclear factor 4alpha was observed earlier in the differentiating cES cells co-cultured with MFLCs, as compared to cES cells undergoing spontaneous differentiation and those subjected to GF-induced differentiation. The expression of cytochrome P450 7a1, a possible marker for embryonic endoderm-derived mature hepatocytes, was only observed in cES cells that had differentiated in a co-culture with MFLCs. Further, the disappearance of Oct3/4, a representative marker of an undifferentiated state, was noted in cells co-cultured with MFLCs, but not in those undergoing spontaneous or GF-induced differentiation. Immunocytochemical analysis revealed an increased ratio of ALB-immunopositive cells among cES cells co-cultured with MFLCs, while glycogen storage and urea synthesis were also demonstrated., Conclusion: MFLCs showed an ability to induce cES cells to differentiate toward hepatocytes. The co-culture system with MFLCs is a useful method for induction of hepatocyte-like cells from undifferentiated cES cells.

Aim: To examine the effects of adiponectin on the functions of Kupffer cells, key modulators of lipopolysaccharide (LPS) -induced liver injury., Methods: D-galactosamine (GalN) and LPS were injected intraperitoneally into adiponectin-/- mice and wild type mice. Kupffer cells, isolated from Sprague-Dawley rats, were preincubated with or without adiponectin, and then treated with LPS., Results: In knockout mice, GalN/LPS injection significantly lowered the survival rate, significantly raised the plasma levels of alanine transaminase and tumor necrosis factor-alpha (TNF-alpha) and significantly reduced IL-10 levels compared with wild type mice. TNF-alpha gene expression in the liver was which higher and those of IL-10 were lower in knockout mice than in wild type mice. In cultured adiponectin-pre-treated Kupffer cells, LPS significantly lowered TNF-alpha levels and raised IL-10 levels in the culture media and their respective gene expression levels, compared with Kupffer cells without adiponectin-pre-treatment., Conclusion: Adiponectin supresses TNF-alpha production and induces IL-10 production by Kupffer cells in response to LPS stimulation, and a lack of adiponectin enhances LPS-induced liver injury.

Hepatitis C virus (HCV) infection is one of the major causes of chronic liver disease worldwide. The critical role of innate as well as adaptive immunity has been reported in HCV persistence and liver injury. In the early phase of acute infection, HCV continues to replicate in the liver, suggesting the HCV capability of inhibiting innate immunity. The sustained, vigorous and multiepitope-specific CD4+ and CD8+ T cell responses are essential for spontaneous HCV clearance. HCV-specific CD8+ T cells are primary elements for HCV clearance by inducing hepatocyte apoptosis, in which Fas/CD95 is fundamentally involved. However, once HCV persistency develops, HCV utilizes multifaceted arms to subvert various immune effectors. During IFNalpha-based therapy, the enhancement of HCV-specific CD4+ T cell response followed by HCV eradication has been reported, however, it remains obscure whether the therapeutic HCV clearance is able to restore the durable immune competency to HCV. Further investigation is still warranted to establish the means to direct HCV-specific immune responses in the desired way.

Concanavalin A (ConA), directly injected into mice, induces T cell-mediated liver injury. However, it remains unclear whether ConA injection can activate innate immune cells, including natural killer (NK) cells and natural killer T (NKT) cells, both of which exist abundantly in the liver. Here we report that ConA injection stimulated interferon (IFN)-gamma production from liver NKT cells as early as 2 hours after injection and augmented YAC-1 cytotoxicity of liver NK cells. ConA-induced NK cell activation required other types of immune cells and critically depended on IFN-gamma. Because a nonhepatotoxic low dose of ConA was capable of fully activating both NKT cells and NK cells, we next addressed the possibility of ConA injection displaying an antitumor effect in the liver without liver injury. A nonhepatotoxic low-dose ConA injection augmented the cytotoxicity of liver NK cells against Colon-26 colon cancer cells and suppressed hepatic metastasis of Colon-26 cells in a NK cell- and IFN-gamma-dependent manner. In conclusion, a nonhepatotoxic low dose of ConA might serve as an immunomodulator that can preferentially activate the innate immune cells to induce an antitumor effect against metastatic liver tumor.

Since hepatocellular carcinomas (HCCs) develop from transformed hepatocytes, sometimes in a multicentrical manner, immunological deletion of such small intrahepatic regions should be an important strategy to prevent HCC development. The liver contains abundant innate cell lineages including natural killer (NK) cells and natural killer T (NKT) cells, the latter of which become activated in a CD1d-restricted manner by alpha-galactosylceramide (alpha-GalCer). In our study, we investigated the anti-tumor effect elicited by alpha-GalCer administration against transplanted hepatoma cells in the liver, in comparison with that in extrahepatic sites. alpha-GalCer administration completely suppressed the growth of BNL 1MEA.7R.1 (BNL) hepatoma cells disseminated in the liver of syngeneic BALB/c mouse but had no anti-tumor effect on subcutaneously implanted BNL cells. Hepatic NKT cells became rapidly activated after alpha-GalCer administration compared to splenic NKT cells and then disappeared. Hepatic NK cells substantially increased their population as well as up-regulated their cytotoxic activity against BNL cells, but NK cells in other tissues, including the spleen, blood and lymph node, did not. Anti-asialo GM1 antibody treatment, which depleted NK cells in vivo, resulted in hepatic tumor formation in alpha-GalCer-treated mice, indicating the critical involvement of NK cells in the alpha-GalCer-induced anti-tumor effect in the liver. In conclusion, our study demonstrates clear differences in NK cell activation and anti-tumor effect through stimulation of NKT cells by alpha-GalCer between the liver and extrahepatic tissues. Sequential activation of these innate cell lineages may be an attractive strategy for controlling micro-disseminated hepatoma cells in the liver., (Copyright 2003 Wiley-Liss, Inc.)

Background: Fulminant non-A, non-B, non-C hepatitis has a high mortality rate, making the identification of its causative agent imperative. Cytomegalovirus, Epstein-Barr virus, human herpesvirus-6, and herpes simplex virus are all members of the herpesviridae family that are associated with fatal hepatic failure. We investigated the involvement of herpesviridae and hepatitis virus in the pathogenesis of fulminant hepatitis., Methods: The study participants consisted of 11 patients with fulminant hepatitis and 11 with acute hepatitis negative for known hepatitis viral markers and any other liver diseases. Viral DNA was extracted from liver tissues and amplified. In situ hybridization was then performed for 1 patient to detect viral DNA and RNA, and viral protein was localized by monoclonal antibodies., Results: Human herpesvirus-6 was detected in liver tissues from seven patients, (five children and two adults) with fulminant hepatitis and two patients with acute hepatitis. Two patients with fulminant hepatitis also had cytomegalovirus in the liver. Although Epstein-Barr virus and herpes simplex virus were detected in the patients with fulminant hepatitis, they were not specific to these patients. In situ hybridization in one of the patients localized DNA and RNA of human herpesvirus-6 in hepatocyte nuclei, and an envelope antigen of this virus was detected in hepatocyte cytoplasm., Conclusions: Human herpesvirus-6 was frequently detected in Japanese pediatric patients with fulminant non-A, non-B, non-C hepatitis. Although the significance of human herpesvirus-6 in liver pathogenesis remains unclear, this virus may replicate in hepatocytes in some patients with acute onset hepatitis.

Recent advances in imaging modalities enable the identification of small hepatocellular nodules. Among the imaging techniques currently used for detecting hepatocellular carcinomas (HCC), computed tomography (CT) during arterial portography (CTAP) is one of the most sensitive techniques available for detecting hemodynamic change. Even so, well-differentiated HCCs that display only limited hemodynamic change, a feature shared with nonmalignant hepatocellular nodules, are not always detectable by CTAP. To improve our ability to distinguish well-differentiated HCCs from nonmalignant hepatocellular nodules, we have attempted to clarify how the characteristics of the nodules are shown by each imaging technique. We studied the imaging and pathological characteristics of 31 nodules (in 22 patients) detected by ultrasonography (US), but not by CTAP. Histological diagnoses were as follows: HCC, 17 of 31 nodules (55%); high-grade dysplastic nodules, 1 of 31 (3%); and nonmalignant nodules, 13 of 31 (42%). Neither digital substraction angiography (DSA) nor CT arteriography (CTA) were able to detect any of the nodules. Detection rates for plain CT were: 5 of 17 (29%) HCC, 1 of 1 (100%) high-grade dysplastic nodules, and 1 of 13 (8%) nonmalignant nodules. Detection rates for T1/T2-weighted magnetic resonance imaging (MRI) were: 4 of 17 (24%) HCC, 1 of 1 (100%) high-grade dysplastic nodules, and 3 of 13 (23%) nonmalignant nodules. Dynamic CT and dynamic MRI provided no additional information. In conclusion, there is some probability that hepatocellular nodules detected by US, but not by CTAP, are HCC. Presently, it is difficult to distinguish between benign nodules and malignant ones with these imaging techniques, and our findings indicate that biopsy may be advisable for nodules detected under these conditions.

Infection with Schistosoma mansoni, a portal vein-residing helminth, is well known to generate life cycle-dependent, systemic immune responses in the host, type 1 deviation during the prepatent period, and type 2 polarization after oviposition. Here we investigated local immunological changes in the liver after infection. Unlike splenocytes, hepatic lymphocytes from infected mice during the prepatent period already produced a higher amount of IL-4 and a lesser amount of IFN-gamma than those from uninfected mice. Hepatic lymphocytes, particularly conventional T cells, but not NK1.1+ T cells, promptly produced IL-4 in response to worm products, soluble worm Ag preparation (SWAP), whenever presented by Kupffer cells from infected mice. The hepatic lymphocytes that had been stimulated with SWAP presented by infected mice-derived Kupffer cells produced a huge amount of IL-4, IL-13, and IL-5 as well as little IFN-gamma in response to immobilized anti-CD3 mAb. Kupffer cells from uninfected mice produced IL-6 and IL-10, but not IL-12 or IL-18, in response to SWAP stimulation and gained the potential to additionally produce IL-4 and IL-13 after the infection. These results suggested that prompt type 2 deviation in the liver after the infection might be due to the alteration of Kupffer cells that induces SWAP-mediated type 2-development of hepatic T cells.

Background & Aims: Fas-mediated apoptosis is one of the major death processes of hepatocytes in liver diseases. The aim of this study was to determine whether interleukin (IL)-1beta regulates the Fas-mediated apoptotic process of differentiated hepatocytes in vivo., Methods: IL-1beta was injected into Balb/cA mice 5 hours before lethal challenge with agonistic anti-Fas administration. Survival and hepatocyte apoptotic process of these mice were examined., Results: IL-1beta pretreatment prolonged animal survival in a dose-dependent manner, and 500 ng of IL-1beta completely protected mice from lethality. Both serum alanine aminotransferase value and hepatic DNA fragmentation were significantly suppressed by IL-1beta pretreatment. IL-1beta affected neither hepatic distribution of anti-Fas antibody nor Fas expression levels on hepatocytes but significantly suppressed Fas-induced activation of hepatic caspase 3-like protease. Suppression of Fas-induced activation of the caspase by IL-1beta was diminished by coadministration with D-galactosamine and reversed by coinjection with an excess amount of uridine., Conclusions: These results suggest that IL-1beta suppresses Fas-mediated hepatocyte apoptosis by inducing molecule(s) that suppress the apoptosis control machinery upstream of caspase 3. This observation raises the possibility that IL-1beta acts as a negative regulator of Fas-mediated hepatocyte apoptosis during liver injury.

Growth factor receptor-bound protein-2 (GRB-2) is a protein linking receptor tyrosine kinase and Sos (Son of Sevenless gene; Ras GDP/GTP exchange protein), leading to activation of the Ras-mitogen-activated protein kinase (MAPK) cascade. So far, it remains unclear how GRB-2 plays a role in signal transduction pathways evoked by hepatotrophic factors. This study was attempted to evaluate the involvement of GRB-2 in signalling in rat hepatocyte growth. Using rat cultured hepatocytes stimulated by hepatotrophic factors and regenerating livers after partial hepatectomy (PH) we examined GRB-2-mediated linkage of hepatotrophic factor receptors to signal transducing molecules such as Sos or dynamin-II by immunoprecipitation and western blot analysis. In primary cultured hepatocytes stimulated with hepatocyte growth factor (HGF) or epidermal growth factor (EGF), GRB-2 linked HGF receptor or EGF receptor, respectively, to Sos which activated the mitogen-activated protein kinase (MAPK) cascade. In contrast, in primary cultured hepatocytes stimulated with insulin, GRB-2 linked insulin receptor substrate-1 (IRS-1) to dynamin-II as well as Sos. In the early phase after PH, GRB-2 activated the Ras-MAPK cascade by linking HGF receptor, IRS-1, or EGF receptor to Sos. In the late phase after PH, a complex of IRS-1-GRB-2 associated with dynamin-II, indicating that GRB-2 may transduce signals from IRS-1 to dynamin-II. We conclude that GRB-2 may play a role in transmitting signals from hepatotrophic factors to not only MAPK but also to other signalling pathways in hepatocyte growth.

Fas-mediated apoptosis is one of the major death processes of hepatocytes in liver diseases. Although compensatory regeneration occurs during liver injury, it has not been determined whether regenerating hepatocytes die by the same apoptotic process as quiescent hepatocytes. To clarify this issue, the hepatocyte apoptotic process, after injection of agonistic anti-mouse Fas, was compared between sham-operated mice and two-thirds partially hepatectomized mice. The onset of hepatocyte apoptosis was retarded in hepatectomized mice, as evidenced by both morphological and biochemical observations, resulting in significantly prolonged animal survival. Flow cytometric analysis revealed similar levels of Fas expression on hepatocytes between hepatectomized mice and sham-operated mice; however, the activation of liver caspase-3-like protease after Fas stimulation was suppressed in hepatectomized mice, whereas pro-caspase-3 expression did not change with or without hepatectomy. Anti-tumor necrosis factor alfa (TNF alpha), when administered before hepatectomy, partially reversed suppression of caspase-3-like activity after Fas stimulation. Furthermore, the injection of TNF alpha into untreated mice suppressed caspase-3-like activity and prolonged animal survival after Fas stimulation. These results indicate that Fas-signaling events at the level or upstream of caspase-3-like protease are suppressed during liver regeneration, resulting in delayed hepatocyte apoptosis, and also that TNF alpha acts as one of the protective factors against Fas-mediated hepatocyte apoptosis.

Objective: To determine the sensitivity of multiple single sections Turbo FLASH MR arterial portography (MRAP) in the detection of hepatic neoplasms., Methods and Patients: Twelve patients with hepatic mass underwent MRAP prior to hepatic resection. Findings of MRAP were compared with surgical specimen and intra-operative ultrasonography (US)., Results: A total of 19 separate malignant neoplastic nodules were identified in the resected specimens or intra-operative ultrasonography. The sensitivity was 89.5% (17/19) for MRAP. MRAP depicted all neoplasms more than 1.0 cm in diameter. Two lesions not depicted on MRAP had a diameter of 5 and 9 mm, respectively. One lesion identified by MRAP was confirmed to false positive lesion by intra-operative US., Conclusion: Multiple single sections Turbo FLASH MRAP may be a valuable adjunct for pre-operative detection of malignant hepatic neoplasms.

When LPS is administered to heat-killed Propionibacterium acnes-primed BALB/c nude mice, they develop endotoxin-induced liver injury. As previously reported, this liver injury can be prevented by treatment with an Ab against IL-18, a novel cytokine with the ability to induce IFN-gamma production and up-regulate functional Fas ligand (FasL) expression. To identify the pathologic role of IL-18 in this liver injury, we investigated the hepatic cytokine network and FasL induction after LPS challenge. After LPS challenge to BALB/c nude mice, their livers expressed IL-12 mRNA, followed by the induction of IFN-gamma and FasL mRNA and then by the late elevation of TNF-alpha mRNA, but stably expressed IL-18 mRNA. The TNF-alpha induction curve had two peaks. The first peak was the result of the direct reaction to LPS, and the late peak might have been induced, since P. acnes-elicited Kupffer cells showed one-peak TNF-alpha kinetics in response to LPS stimulation in vitro. LPS-activated P. acnes-elicited Kupffer cells secreted both IL-12 and IL-18, as determined by ELISA and bioassay, respectively. The in vivo administration of anti-IL-18 just before an LPS challenge suppressed not only the induction of IFN-gamma and the late TNF-alpha elevation, but also the FasL induction, resulting in the total prevention of liver injury, whereas such an anti-IL-12 treatment did not. Anti-IFN-gamma treatment reduced the late increase in TNF-alpha, but not FasL, resulting in a partial prevention of the liver injury. The administration of anti-TNF-alpha just before elevation of the late TNF-alpha peak also markedly, but incompletely, suppressed the LPS-induced liver injury. These data suggested that IL-18 activates both TNF-alpha- and FasL-mediated hepatocytotoxic pathways in endotoxin-induced liver injury.

LPS injection into normal mice does not induce liver injury, while the same treatment of Propionibacterium acnes-primed mice induces severe liver injury, indicating that P. acnes treatment renders the mice susceptible to LPS. Since IFN-gamma sensitizes macrophages to LPS, we investigated the mechanism of induction and activation of IFN-gamma-producing (type 1) T cells by P. acnes. Twenty percent of liver lymphocytes of C57BL/6 mice are CD4+ NK1.1+ T cells that promptly produce IL-4 in response to anti-CD3 in vitro. However, P. acnes treatment diminished these lymphocytes. Therefore, liver lymphocytes from P. acnes-primed mice showed reduced IL-4 production. Furthermore, P. acnes treatment induced CD4- type 1 T cells in the liver. Isolated P. acnes-elicited Kupffer cells produced IL-12 and to a lesser degree IL-18 in vitro. Injection of anti-IL-12 Ab totally abrogated these actions of P. acnes, while injection of anti-IL-18 Ab caused only partial abrogation. Thus, administration of P. acnes diminished CD4+ NK1.1+ T cells, but induced type 1 T cells in the liver by induction of IL-12 and IL-18 production. Injection of IL-12 (approximately 1,000 ng) dose dependently diminished CD4+ NK1.1+ T cells, but induced type 1 T cells. In contrast, injection of IL-18 (approximately 1,000 ng) failed, although injection of a much larger dose of IL-18 (10,000 ng) or IL-18 (approximately 1,000 ng) with suboptimal doses of IL-12 (1-100 ng) diminished CD4+ NK1.1+ T cells in a dose-dependent manner. Thus, P. acnes treatment renders the mice highly susceptible to LPS by induction and activation of type 1 T cells.

We recently reported that transrectal or intestinal portal scintigraphy with 123I-iodoamphetamine (IMP) could be a useful method for the non-invasive and quantitative evaluation of the portosystemic shunt in portal hypertension, but what cells in the liver trap IMP has not been clarified. This study was aimed at elucidating whether IMP was extracted by parenchymal cells, sinusoidal endothelial cells, Kupffer cells or fat storing cells. Each type of liver cell was isolated from rats and cultured. The cells were incubated with 125I-IMP and the radioactivity of the lysate was determined. Nonspecific binding was assessed in the presence of an excess of unlabeled IMP, and specific binding was determined by subtracting the nonspecific from total binding. Specific binding observed in parenchymal cells, endothelial cells and Kupffer cells was 70.2 +/- 0.4, 4.2 +/- 1.4 and 2.3 +/- 0.8 pmol/well, respectively, but no specific binding was observed in fat storing cells. The binding in parenchymal cells was much higher than that in endothelial cells or Kupffer cells (p < 0.005). In addition, the binding to parenchymal cells reached equilibrium within 20 min and was not saturable over the concentration range tested (0.5-10 microM). These findings indicate that IMP is mostly extracted by parenchymal cells in the liver.

The distinctive histologic findings in acute hepatitis A, B, and C suggest that different immunological mechanisms are involved in the pathogenesis of these diseases. This study was undertaken to define the immune response in each type of acute hepatitis by identification of the intrahepatic lymphocyte subsets. Thirty paraffin-embedded liver biopsy specimens from 10 patients with acute hepatitis A, 10 patients with acute hepatitis B, and 10 patients with acute hepatitis C were evaluated. Immunohistochemical staining was performed by the indirect immunoperoxidase technique using the following monoclonal or polyclonal antibodies: CD45RO, CD20-cy, CD57, and Mac387. Inflammatory infiltrates varied from specimen to specimen. However, CD45RO+ memory T cells were the predominant infiltrating mononuclear cells in all specimens. In the portal areas, CD45RO+ memory T cells were the most prominent in AHC, followed by AHA and AHB, and the difference between AHC and AHB was statistically significant. CD20-cy+ B cells were seen mainly in the portal areas, and were significantly less common in AHB than in AHA and AHC. In addition, the ratio of CD20-cy+ B cells to CD45RO+ memory was significantly lower in AHB than in the other types of acute hepatitis. The necrotic areas in all specimens contained mainly CD45RO+ memory cells in association with a few CD57+ NK and T cells or CD20-cy+ B cells. Our study revealed differences of the intrahepatic lymphocyte subsets among the various types of acute hepatitis, but the meaning of these differences is presently unknown. Therefore, further studies are required to fully elucidate the mechanism of the immune response in acute hepatitis.

Background: Hepatitis B virus-infected cells can be eliminated by the cytotoxic T cell-mediated immune reaction. Fas ligand, recently detected on the surface of cytotoxic T cell, is thought to induce cells to apoptosis by adhering to Fas antigen., Aims/methods: To evaluate the role of Fas antigen and apoptosis in chronic hepatitis B, we immunohistochemically studied Fas antigen and HBsAg expression in liver samples from patients with hepatitis B virus infection., Results: In samples from 56 HBV patients, Fas antigen was mainly expressed in the cytoplasm (partly at the membrane) of hepatocytes, and these positive cells were detected especially at the periportal region near "piecemeal necrosis". According to Knodell's HAI scoring system, the scores of periportal inflammation and necrosis (category I) and the scores of intralobular inflammation and necrosis (category II) were similarly higher in Fas antigen-positive cases than in Fas antigen-negative cases (p < 0.01), and there was a positive correlation between these scores and the degree of Fas antigen expression. In normal cases, Fas antigen was not detected. In patients with HBV infection, Fas antigen expression was closely correlated with the activity of the viral hepatitis. HBsAg was expressed by the majority of hepatocytes. However, Fas antigen was expressed by fewer hepatocytes than the number of HBsAg-positive cells., Conclusions: These findings suggest that the expression of Fas antigen may not be triggered only by HBV infection, and immunological interaction may be needed for the expression and for apoptosis to occur.

The purpose of this study was to examine the effect of different amounts of fat in enteral diets on liver function, liver lipid accumulation, and protein metabolism in septic rats. Sepsis was induced in Wistar rats by cecal ligation and puncture. The rats were divided into four groups and were fed enterally 0% (FO, n = 7), 10% (F10, n = 7), 20% (F20, n = 8), or 30% (F30, n = 9) of total calories as fat. The liquid diet consisted of medium-chain and long-chain triglyceride mixtures as the fat sources, casein oligopeptide, and dextrin (100 kcal/100 ml). Intraduodenum feeding was ended on the 6th day. Serum glutamic oxaloacetic transaminase and glutamic pyruvic transaminase activities, indices of liver dysfunction, were highest in the FO group, and triglycerides accumulated in the livers of that group, possibly because of the large proportion of carbohydrate in the diet. Value of nitrogen balance was highest in the F 10 group, and serum total protein and albumin concentration were higher in the F10 and F20 groups than in the FO and F30 groups. The liver protein content in the F10 and F20 groups was higher than in the FO and F30 groups. Serum triglyceride in the F30 group was about 2 times higher than in the F10 and F20 groups. These results indicate that enteral fat administration in septic rats as 30% of total calories reduced liver dysfunction and liver triglyceride accumulation, but might have been excessive for optimal protein metabolism. Therefore, the preferable amount may range from 10% to 20% of total calories.

An in vivo model of hepatitis C virus (HCV) infection is needed to enable investigation of the mechanism of the liver injury that it causes. In this study, we used asialoglycoprotein receptor mediated gene delivery to obtain expression of the complementary DNA (cDNA) coding the core and part of the envelope 1 protein of HCV because selective delivery to the hepatocytes has been reported to be attained with this method. The optimum carrier-DNA ratio was examined using in vitro transfection and found to be important for the efficiency of this method. In transfection in vivo, microautoradiographical examination showed that the transfected plasmids were delivered selectively to the liver parenchymal cells. To obtain an immunohistochemically detectable level of protein expression in rodent liver, some modifications for increasing the in vivo transfection efficiency were performed; a lysosomal enzyme inhibitor, chloroquine, was used and the administration route of the carrier-DNA complex was changed from the tail vein to the portal vein. On the bases of these results, in vivo transfection with expression vector of HCV core/E1 region was performed. In rat liver transfected by intraportal injection with chloroquine, the transcript RNA and the core protein were detected. These results indicated that the HCV core/E1 expression vector was not merely delivered but also successfully expressed in the liver using asialoglycoprotein receptor mediated gene delivery. The number of the HCV core expressing cells in the transfected liver was similar to that in patients with hepatitis C. These in vivo transfected animals should be useful for investigating the role of this region in the liver injury caused by HCV.

Hepatitis C virus (HCV) has been reported to conform to a quasispecies nature, which is most evident in hypervariable regions of the putative envelope 2 domain. The aim of this study was to determine the relationship between the nucleotide complexity and diversity of hypervariable region 1 and various stages of the carrier states. The subjects studied were 20 HCV carriers with normal alanine aminotransferase (ALT) levels, 50 patients with chronic hepatitis who showed elevated ALT levels, 22 with cirrhosis, and 24 with hepatocellular carcinoma. The quasispecies complexity was analyzed by means of polymerase chain reaction-mediated single strand conformation polymorphism (PCR-SSCP). The value of nucleotide diversity was calculated by PCR cloning and sequencing. The number of SSCP bands ranged from 1 to 7, with no significant differences in the mean numbers among the stages of HCV infection. There was no correlation between the amounts of serum HCV RNA and the numbers of SSCP bands. No significant difference was found in the values of nucleotide diversity between carriers with normal ALT levels (mean, 6.6 x 10(-2) per site) and patients with chronic hepatitis (7.7 x 10(-2). These findings suggest that the quasispecies complexity of hypervariable region 1 is independent of the stage of chronic HCV infection.

The effect of the hepatocyte growth factor (HGF) on the Na+/H+ exchanger was studied using primary cultured hepatocytes. HGF induced intracellular pH (pHi) elevation of 0.10 pH units in hepatocytes cultured for 4 to 7 hours; the response was lower after other culture periods. Even with the same culture period, intercellular heterogeneity was found in the responsiveness to HGF. This heterogeneity may be partially accounted for by the weak but significant correlation observed between the basal pHi level and the degree of pHi elevation caused by HGF in hepatocytes. The pHi elevation caused by HGF was blocked on pretreatment of the hepatocytes with amiloride, suggesting that HGF activates the Na+/H+ exchanger. This hypothesis was confirmed by the fact that HGF increased the initial rapid rate of cell alkalization of acid-loaded hepatocytes. The tyrosine kinase inhibitor, genistein, also blocked the elevation, consistent with the fact that HGF receptor/c-met has a tyrosine kinase domain. To clarify the signal transduction pathway from tyrosine kinase to the Na+/H+ exchanger, we examined the effects of inhibitors of other kinases (H-7, H-8, and W-7) on the HGF-induced pHi elevation and found that only W-7 blocked it. This pHi elevation was also prevented on preincubation of the hepatocytes with thapsigargin, which blocks the calcium response caused by HGF. These results suggest that HGF activates the Na+/H+ exchanger in hepatocytes through a tyrosine kinase-calcium/calmodulin-dependent pathway.

The lack of a small animal model of hepatitis C virus (HCV) infection has impeded elucidation of the pathogenesis of HCV. The aim of this study was to develop an HCV-expressing animal model by means of cationic liposome-mediated in vivo gene transfer. To examine the feasibility of this strategy, pActLacZ, an expression vector composed of the LacZ gene driven by the beta-actin promoter, complexed with lipofectin, was injected retrogradely into the common bile ducts of adult rats. X-Gal histochemical staining clearly showed that the LacZ gene was expressed in hepatocytes, but not in biliary epithelial cells. Maximal expression was observed at a DNA to lipofectin ratio of 1:4. Based on this observation, pAGS3M091, an expression vector containing the full length of HCV complementary DNA (cDNA) preceded by the beta-actin promoter, was evaluated. Two days after in vivo intrabiliary administration of pAGS3M091 complexed with lipofectin, polymerase chain reaction (PCR) amplification of reverse-transcribed liver RNA demonstrated the 5' and 3' portions of HCV transcripts derived from pAGS3M091. Immunohistochemical analysis showed the HCV core protein in a small number of hepatocytes scattered in the hepatic lobules. We conclude that the full-length HCV genome was successfully expressed in adult rat liver by means of cationic liposome-mediated in vivo gene transfer. This model will be useful for determining the immunopathological role of HCV in vivo.

The lack of a small animal model for hepatitis C virus (HCV) infection has impeded elucidation of the pathogenesis of this virus. The aim of this study was to develop an HCV-expressing animal model using cationic liposome-mediated in vivo gene transfer. To examine the feasibility of this strategy, an expression vector composed of the LacZ gene driven by the beta-actin promoter, pActLacZ, was injected retrogradely into the common bile ducts of adult rats. X-Gal histochemical staining clearly showed that the LacZ gene was expressed in hepatocytes. Maximal expression was observed at a DNA:lipofectin ratio of 1:4. Based on this observation, an expression vector containing the full-length of HCV cDNA, pAGS3M091, was evaluated in adult rats. Two days after intrabiliary administration of pAGS3M091, PCR amplification of reverse-transcribed liver RNA demonstrated the 5' and 3' portions of HCV transcripts derived from pAGS3M091. Immunohistochemical analysis revealed the HCV core protein in a small number of hepatocytes scattered in the lobules. Thus, the full-length of the HCV genome was successfully expressed in adult rat liver using liposome-mediated in vivo gene transfer.

To investigate the signaling pathways to Na+/H+ exchanger activation with epidermal growth factor in hepatocytes, we measured changes in cytosolic free calcium and intracellular pH levels at the single-cell level using digital imaging fluorescence microscopy of fura-2- or BCECF-loaded hepatocytes in primary culture. Epidermal growth factor induced cytosolic free calcium oscillations consisting of periodic trains of spikes with a latency period of up to several minutes. These calcium responses were inhibited by tyrosine kinase inhibitor genistein (100 mumol/L) and abolished by emptying of intracellular Ca2+ pools with 3 mumol/L thapsigargin, an inhibitor of Ca(2+)-ATPase on the endoplasmic reticulum. Epidermal growth factor (1 nmol/L) induced an intracellular pH increase of 0.12 +/- 0.07 units from the basal level of 7.25 +/- 0.09 units after several minutes of latency. This effect was completely abolished by 1 mmol/L amiloride, an inhibitor of the Na+/H+ exchanger. The epidermal growth factor-induced intracellular pH increase was inhibited by pretreatment of hepatocytes with genistein (100 mumol/L), thapsigargin (3 mumol/L) or calmodulin inhibitor W-7 (25 mumol/L), but not with protein kinase C inhibitor H-7 (50 mumol/L) or with cyclic AMP-dependent kinase inhibitor H-8 (60 mumol/L). Phorbol ester PMA (phorbol 12-myristate 13-acetate), a potent activator of protein kinase C, induced a slight intracellular pH increase significantly smaller than that with epidermal growth factor, whereas this effect was completely blocked by pretreatment with H-7, indicating that PMA-induced intracellular pH increase is mediated by protein kinase C pathways, unlike epidermal growth factor.(ABSTRACT TRUNCATED AT 250 WORDS)

Objectives: Liver histology in volunteer blood donors positive for serum hepatitis C virus RNA was investigated in relation to hepatitis C virus viremia levels., Methods: Twenty-one volunteer blood donors positive for serum hepatitis C virus RNA by polymerase chain reaction were monitored for at least 1 yr by monthly routine liver function tests and underwent liver biopsy. Liver histology findings were correlated with hepatitis C virus viremia levels assessed by a quantitative branched DNA assay., Results: Liver histology showed the features of chronic hepatitis in 20 (95%) patients. Only one of the seven patients with persistently normal aminotransferase levels during follow-up had normal liver histology, and the others had chronic hepatitis. Sera obtained the same day of the liver biopsy were shown to contain hepatitis C virus RNA of 10(5.7)-10(7.6) equivalent/ml (median 10(6.7)). The total histological activity index score (median 2, range 0-15) and the scores of portal inflammation (median 1, range 0-3), lobular inflammation (median 1, range 0-4) and piecemeal necrosis (median 0, range 0-5) correlated with viremia levels (r = 0.64, p < 0.01; r = 0.60, p < 0.01; r = 0.48, p < 0.05; and r = 0.49, p < 0.05, respectively)., Conclusions: These findings suggest that chronic hepatitis is frequently caused by hepatitis C virus infection irrespective of the serum aminotransferase levels, and high level hepatitis C virus replication is a contributory cause for liver injury in volunteer blood donor populations.