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Overt hepatic encephalopathy (HE) is one of the complications of liver cirrhosis (LC), which negatively affects the prognosis and quality of life of patients. Small intestinal bacterial overgrowth (SIBO) is significantly associated with LC and its complications, including HE. We investigated the relationship between SIBO and LC, and the difference between hydrogen-producing and methane-producing SIBO (H-SIBO and M-SIBO, respectively). This is a prospective cohort study of 107 cases. Breath measurements of hydrogen and methane concentrations were performed for the diagnosis of SIBO. The study cohort included 81 males with a median age of 70 (40-86) years, and SIBO was detected in 31 cases (29.0%). There were no significant differences between the SIBO positive and SIBO negative groups. Reclassification into H-SIBO (16 cases) and others (91 cases) was performed, and the Child-Pugh score was only derived in the multivariate logistic analysis (P = 0.028, odds ratio 1.39, 95% confidence interval 1.04-1.85). Furthermore, H-SIBO was significantly associated with covert HE in chi-square test (50.0% vs. 24.2%, P = 0.034). In addition, we evaluated the therapeutic response on SIBO of rifaximin in eight covert HE patients. 20% patients with M-SIBO and 67% patients with H-SIBO showed an improvement of the breath test. In conclusion, H-SIBO, but not M-SIBO, is significantly associated with liver function, and rifaximin might be more effective for covert HE with H-SIBO. Therefore, the diagnosis of SIBO, including the classification as H-SIBO and M-SIBO, might help to determine the choice of treatment for HE., Competing Interests: This work was supported by research grants from ASKA Pharmaceutical Co., Ltd. and the Japan Agency for Medical Research and Development. In addition, Shuji Terai received a lecture fee from ASKA Pharmaceutical Co., Ltd, Japan. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Low-energy extracorporeal shock waves (LESW) have been studied as a new treatment for angina pectoris and several ischemic diseases because of its effect on angiogenesis and inhibition of fibrosis of the heart. The effect of LESW on fibrosis in liver cirrhosis has not been studied. The aim of this study was to verify the amelioration of liver fibrosis by LESW and elucidate its mechanisms in a rat model of drug-induced liver cirrhosis. Male Wistar rats aged 7 weeks were injected with carbon tetrachloride intraperitoneally twice a week for 12 weeks. Eight rats underwent LESW therapy (0.25 mJ/mm 2 , 4 Hz, 1000 shots) under general anesthesia (shock wave group). Seven rats only underwent general anesthesia (control group). Quantitative analysis showed that the area of fibrosis in the shock wave group was significantly reduced compared with the control group (11,899.9 vs. 23,525.3 pixels per field, p < 0.001). In the shock wave group, the mRNA expression of transforming growth factor (TGF)-β1 was significantly suppressed (0.40-fold, p = 0.018) and vascular endothelial growth factor-B was significantly increased (1.77-fold, p = 0.006) compared with the control group. Serum albumin was significantly higher in the shock wave group than in the control group (3.0 vs. 2.4 g/dl, p = 0.025). Aspartate aminotransferase/alanine aminotransferase ratio decreased by LESW compared with the control group (1.49 vs. 2.04, p = 0.013). These results suggest that LESW therapy ameliorates liver fibrosis by reducing the expression of TGF-β1 and increasing the expression of angiogenic factors, and improves hepatic function.

Background: Preservation of the hepatic vessels while dividing the parenchyma is key to achieving safe liver resection in a timely manner. In this study, we assessed the feasibility of a newly developed, piezo actuator-driven pulsed water jet (ADPJ) for liver resection in a surviving swine model., Methods: Ten domestic pigs underwent liver resection. Parenchymal transection and vessel skeletonization were performed using the ADPJ (group A, n = 5) or an ultrasonic aspirator (group U, n = 5). The water jet was applied at a frequency of 400 Hz and a driving voltage of 80 V. Physiological saline was supplied at a flow rate of 7 ml/min. After 7 days, the animals were killed and their short-term complications were examined and compared between the two groups., Results: No significant complications, such as massive bleeding, occurred in either group during the surgical procedures. The transection time per transection area was significantly shorter in group A than in group U (1.5 ± 0.3 vs. 2.3 ± 0.5 min/cm(2), respectively, P = 0.03). Blood loss per transection area was not significantly different between groups A and U (9.3 ± 4.2 vs. 11.7 ± 2.3 ml/cm(2), P = 0.6). All pigs in group A survived for 7 days. No postoperative bleeding or bile leakage was observed in any animal at necropsy., Conclusion: The present results suggested that the ADPJ reduces transection time without increasing blood loss. ADPJ is a safe and feasible device for liver parenchymal transection.

We have previously reported that epiregulin is a growth factor that seems to act on liver progenitor cells (LPCs) during liver regeneration. However, the relationship between epiregulin and LPCs has remained unclear. The aim of the present study was to clarify the role of epiregulin during liver regeneration. The serum levels of epiregulin in patients with acute liver failure were examined. A liver injury model was developed using mice fed a diet containing 0.1% 3.5-diethoxycarbonyl-1.4-dihydrocollidine (DDC) to induce LPCs. We then evaluated the expression of epiregulin and LPCs in these mice. The proliferation of epithelial cell adhesion molecule + LPCs cultured with epiregulin was examined in vitro, and finally epiregulin was overexpressed in mouse liver. In patients with acute liver failure, serum epiregulin levels were elevated significantly. In DDC mice, LPCs emerged around the portal area. Epiregulin was also detected around the portal area during the course of DDC-induced liver injury and was partially coexpressed with Thy1. Serum epiregulin levels in DDC mice were also significantly elevated. Recombinant epiregulin augmented the proliferative capacity of the LPCs in a dose-dependent manner. In mice showing overexpression of epiregulin, the expression of PCNA on hepatocytes was increased significantly. Finally, LPCs emerged around the portal area after epiregulin gene delivery. We concluded that epiregulin promotes the proliferation of LPCs and DNA synthesis by hepatocytes and is upregulated in the serum of patients with liver injury. Furthermore, induction of epiregulin leads to the appearance of LPCs. Epiregulin would be a useful biomarker of liver regeneration., (Copyright © 2014 the American Physiological Society.)

Background/purpose: The water jet technique dissects tissue while sparing cord-like structures such as blood vessels. The mechanism of such tissue-selective dissection has been unknown. The novel piezo actuator-driven pulsed water jet (ADPJ) system can achieve dissection with remarkably reduced water consumption compared to the conventional water jet; however, the system's characteristics and dissection capabilities on any organ have not been clarified. The purposes of this study were to characterize the physical properties of the novel ADPJ system, evaluate the dissection ability in swine organs, and reveal the mechanism of tissue-selective dissection., Methods: The pulsed water jet system comprised a pump chamber driven by a piezo actuator, a stainless steel tube, and a nozzle. The peak pressure of the pulsed water jet was measured through a sensing hole using a pressure sensor. The pulsed water jet technique was applied on swine liver in order to dissect tissue on a moving table using one-way linear ejection at a constant speed. The dissection depth was measured with light microscopy and evaluated histologically. The physical properties of swine liver were evaluated by breaking strength tests using tabletop universal testing instruments. The liver parenchyma was also cut with three currently available surgical devices to compare the histological findings., Results: The peak pressure of the pulsed water jet positively correlated with the input voltage (R(2) = 0.9982, p < 0.0001), and this was reflected in the dissection depth. The dissection depth negatively correlated with the breaking strength of the liver parenchyma (R(2) = 0.6694, p < 0.0001). The average breaking strengths of the liver parenchyma, hepatic veins, and Glisson's sheaths were 1.41 ± 0.45, 8.66 ± 1.70, and 29.6 ± 11.0 MPa, respectively. The breaking strength of the liver parenchyma was significantly lower than that of the hepatic veins and Glisson's sheaths. Histological staining confirmed that the liver parenchyma was selectively dissected, preserving the hepatic veins and Glisson's sheaths in contrast to what is commonly observed with electrocautery or ultrasonic instruments., Conclusions: The dissection depth of liver tissue is well controlled by input voltage and is influenced by the moving velocity and the physical properties of the organ. We showed that the device can be used to assure liver resection with tissue selectivity due to tissue-specific physical properties. Although this study uses an excised organ, further in vivo studies are necessary. The present work demonstrates that this device may function as an alternative tool for surgery due to its good controllability of the dissection depth and ability of tissue selectivity.

Many patients with biliary atresia (BA) have impaired metabolism of copper (Cu) and zinc (Zn) because of the obstruction of bile ducts. An excessive Cu accumulation is cytotoxic and results in fibrosis in hepatic tissues. Since Zn works antagonistically to Cu, lower Zn concentrations may deteriorate liver damage. In the 1980's, we performed a series of surgeries on BA patients for the construction and alteration of the bile flow route, which is the major excretion route for Cu. We obtained liver and serum samples at each surgery, and measured Cu and Zn concentrations by inductively coupled plasma atomic emission spectrometry. Hepatic Cu concentration decreased with the improvement of cholestasis after the establishment of bile excretion. Conversely, when cholestasis persisted or recurred, increases in hepatic and serum Cu concentrations were noted. Hepatic Zn concentration was lower than previously reported normal values. High hepatic and serum Cu concentrations due to persistent or recurrent cholestasis and low hepatic Zn concentration may deteriorate hepatic fibrosis and liver cirrhosis.

Human umbilical cord blood (UCB) cells have many advantages as grafts for cell transplantation because of the immaturity of newborn cells compared with adult cells. In contrast to their hematopoietic and mesenchymal potential, it remains unclear whether UCB cells have endodermal competence. Here, with a view to utilize UCB cells for cell transplantation into injured liver, we investigated the hepatic potential of UCB cells both in vitro and in vivo. We determined the most efficient conditions leading UCB cells to produce albumin (ALB). In a novel primary culture system supplemented with a combination of growth/differentiation factors, about 50% of UCB cells in 21-day cultures expressed ALB, and the ALB(+) cells coexpressed hepatocyte lineage markers. The ALB-expressing cells were able to proliferate in the culture system. Moreover, in the cell-transplantation model into liver-injured severe combined immunodeficient mice, inoculated UCB cells developed into functional hepatocytes in the liver, which released human ALB into the sera of the recipient mice. In conclusion, this study demonstrates that human UCB is a source of transplantable hepatic progenitor cells. Our findings may have relevance to clinical application of UCB-derived cell transplantation as a novel therapeutic option for liver failure.

Objective: To clarify gene expression profiles in the liver may elucidate the pathogenesis of type I autoimmune hepatitis (AIH). Using suppression subtractive hybridization (SSH), we identified genes overexpressed in the liver of AIH., Methods: A small liver biopsy sample from a patient with definite AIH was available to be analyzed in our system. By mixing cDNA synthesized from this sample as a 'tester' and cDNA from a normal liver as a 'driver,' we subtracted cDNA to enrich genes overexpressed in AIH. After polymerase chain reaction (PCR) amplification and subcloning, we identified subtracted genes by sequencing 50 randomly selected clones., Results: Only one cDNA fragment, which is identical to interferon inducible protein 10 (IP-10), was overexpressed by > 10 times in the liver of AIH, as compared with control. We confirmed IP-10 overexpression in all eight patients with AIH by reverse transcription PCR. Immunohistochemical analysis demonstrated increased IP-10 expression in hepatocytes in the liver of AIH. Reverse transcription PCR analysis of 63 liver biopsy samples with various liver diseases revealed that IP-10 expression was significantly higher in AIH (p = 0.025) and chronic hepatitis C (p = 0.0043) than in other liver diseases. Interestingly, the amount of IP-10 mRNA expression was correlated with serum ALT values in AIH (p = 0.0006), but not in chronic hepatitis C (p = 0.43)., Conclusion: These results indicate the IP-10 expression in the liver might be used as a preferential marker of AIH, and that IP-10 has some pathophysiological roles in the liver damage of AIH.

Background and Aim: It has been a conflicting issue whether TT virus (TTV), a newly isolated DNA virus from a patient with liver injury of unknown cause, is a causative agent of acute and/or chronic hepatitis. TT Virus DNA titers were shown to be 10-100-fold greater in liver tissue than in serum, whereas the majority of TTV-positive cases had no biochemical or histological evidence of significant liver damage. We therefore attempted in situ hybridization to investigate whether TTV is hepatotropic., Methods: Because of the marked divergence in TTV genome types, a template for TTV-DNA (coding region for N22 clone) was amplified and labeled with digoxigenin-dUTP by using hemi-nested PCR from the serum, then DNA probes were applied to the liver sections of the same case. After hybridization, the probes were visualized immunohistochemically. Besides TTV-DNA-negative cases, competitive inhibition experiments with unlabeled probes were performed to confirm the specificity., Results: There were no positive signals in the negative controls, and the intensity of positive signals was markedly diminished in the competitive inhibition experiments. No cross-hybridization with different genotype probes also confirms the specificity. Under the optimal conditions, the positive signals were located in the cytoplasm of the hepatocytes in eight of nine TTV-DNA-positive cases. The signals were not seen in non-parenchymal cells of the liver., Conclusion: TT Virus is proved to be hepatotropic by in situ hybridization.

As a part of the defense mechanism of the host to viral infection, interferons induce the transcription of several genes. These interferon-inducible genes contribute to the eradication of the viruses. Whereas some studies suggested the participation of a dsRNA-dependent protein kinase in the host reaction to hepatitis C virus infection, the involvement of other interferon-inducible genes has not been evaluated. Furthermore, there has been no analysis on the expression profile of multiple interferon-inducible genes. The aim of this study was to clarify the hepatic mRNA expression profile of interferon-inducible genes with a special concern to chronic hepatitis C. A total of 76 liver biopsy samples (28 with chronic hepatitis C, 10 with chronic hepatitis B, 9 with alcoholic liver disease, 14 with autoimmune hepatitis, 10 with primary biliary cirrhosis, and 5 of normal liver) were enrolled. The expression of the following genes was quantified by competitive reverse transcription-polymerase chain reaction and was compared according to the etiology; dsRNA-dependent protein kinase (PKR), 2',5'-oligoadenylate synthetase (2,5-AS), latent cellular endoribonuclease (RNase L), RNase L inhibitor, and MxA. As a result, PKR mRNA was significantly overexpressed in the liver of chronic hepatitis C compared with those of other etiologies (P =.0178), and it correlated significantly with serum alanine transaminase values (r =.51, P =.0054). Also, the expression of the RNase L inhibitor showed a significant reduction in chronic hepatitis C (P =.0184). The expressions of 2,5-AS, RNase L, and MxA were not different significantly irrespective to the etiology. In conclusion, hepatic overexpression of PKR and reduced expression of RNase L inhibitor seem to contribute to the anti-HCV mechanism characteristically.

To examine whether adenosine reduces ischemia/reperfusion (I/R)-induced liver injury by inhibiting leukocyte activation via A(2) receptor (A(2)R) stimulation, we investigated the effects of adenosine and selective A(2A) receptor (A(2A)R) agonists (YT-146 and CGS21680C) on I/R-induced liver injury in rats. Adenosine, YT-146, and CGS21680C, in the concentration of 10(-7) to 10(-5) M, significantly inhibited neutrophil elastase release by about 30 to 40% and increased intracellular Ca(2+) concentrations in isolated neutrophils stimulated with formyl-methionyl-leucyl-phenylalanine (fMLP) in vitro. Adenosine, YT-146, and CGS21680C, in the concentration of 10(-7) to 10(-5) M, significantly inhibited tumor necrosis factor (TNF)-alpha production by monocytes stimulated with endotoxin by about 50%. Although ZM241385, a selective A(2A)R antagonist, significantly enhanced the increase in neutrophil elastase release and intracellular Ca(2+) concentrations in neutrophils stimulated with fMLP, this agent did not affect the endotoxin-induced TNF-alpha production by monocytes. Rats were subjected to liver ischemia for 60 min. Serum levels of transaminases increased after hepatic I/R, peaking at 12 h after reperfusion. The i.v. infusion of adenosine (1 and 10 mg/kg/h), YT-146 (0.1 and 1 mg/kg/h), and CGS21680C (0.1 and 1 mg/kg/h) significantly inhibited the I/R-induced increase in serum transaminase levels 12 h after reperfusion. The I/R-induced decrease in hepatic tissue blood flow was significantly prevented by adenosine and YT-146. Hepatic levels of TNF-alpha, cytokine-induced neutrophil chemoattractant (equivalent to human interleukin-8), and myeloperoxidase were significantly increased after I/R. These increases were significantly inhibited by the administration of adenosine, YT-146, and CGS21680C. Although the histological neutrophil accumulation in the liver was significantly increased after I/R as evaluated by the naphthol AS-D chloroacetate technique, the administration of adenosine, YT-146, and CGS21680C significantly inhibited this increase. These findings suggest that adenosine reduces I/R-induced liver injury both by inhibiting the synthesis of inflammatory mediators and by inhibiting neutrophil degranulation directly, probably through A(2A)R stimulation.

Inflammation of the portal and periportal areas is a common feature of chronic hepatitis C. Antigen-presenting dendritic cells are located in the portal area, and infiltrating T cells are initially exposed to infected hepatocytes in the periportal area. Thus, these areas could be sites of the initial processes of the immune response in chronic hepatitis C. C-C chemokines (dendritic-cell-derived C-C chemokine [DC-CK1] and regulated upon activation, normal T-cell expressed and secreted [RANTES])-attracting T cells may play a role in the portal inflammatory changes. The relationship between the expression of these C-C chemokines, which attract T cells and the infiltration of T cells into the liver of patients with chronic hepatitis C, was examined by in situ hybridization and reverse transcription-polymerase chain reaction. T-cell activation was examined by immunostaining T-cell subsets. Specific signals were detected for DC-CK1 mRNA in mononuclear cells mainly in the portal area and for RANTES mRNA in the portal area and at sites of piecemeal necrosis in the liver of patients with chronic hepatitis C. Naive T cells were located mainly in the portal area, whereas active T cells were found mainly at sites of piecemeal necrosis in the periportal area. In addition, hepatic DC-CK1- and RANTES-mRNA levels were significantly correlated with serum alanine aminotransferase levels (p < 0.001). These results suggest that the local production of DC-CK1 and RANTES participates in immune responses by attracting naive and active T cells to the portal and periportal areas, respectively.

To study the effects of percutaneous ethanol injection therapy on livers, we investigated necrotic changes after ethanol injection and fibrotic changes during the repair process in cirrhotic livers in comparison with normal livers. Male rats were treated with oral doses of 3'-methyl-4-dimethylaminoazobenzene and thioacetamide to produce liver cirrhosis. Both control animals and cirrhotic animals were injected with 0.2 ml of absolute ethanol into livers. Histological samples were cut serially, and the maximum areas of necrosis and fibrosis were measured until 28 days after the injection. Although the maximum area of necrosis was not different between cirrhotic livers and control livers, the average fibrotic ratio [(maximum fibrotic area/maximum necrotic area + maximum fibrotic area) x 100] was 64% in control livers (n = 9) and 40% in cirrhotic livers (n = 9; p < 0.05). The fibrotic repair process after ethanol injection seems to be impaired in cirrhotic livers as compared with normal livers.

O6-methylguanine DNA methyltransferase (MGMT) is a repair protein that transfers methyl groups from O6-methylguanine to a cysteine acceptor in its own molecule, and restores DNA to its undamaged state. If left unrepaired, O6-methylguanine can pair with either a thymine or a cytosine, causing a C-G to T-A transition, which is considered to be one of the molecular mechanisms of both mutagenesis and carcinogenesis. The expression of MGMT mRNA in liver tissue was quantitatively assessed by the competitive reverse transcription-polymerase chain reaction method in patients with chronic liver diseases with or without alcohol drinking. MGMT mRNA expression was 1.4 +/- 0.9 pg/micrograms RNA in control livers. Its expression in chronic hepatitis was 3.8 +/- 0.7 in alcoholics and 2.7 +/- 0.8 in nonalcoholics, which were not statistically different. MGMT mRNA expression in liver cirrhosis was significantly low, compared with that in chronic hepatitis, and 0.8 +/- 0.3 in alcoholics and 0.5 +/- 0.1 in nonalcoholics, which also were not significantly different. The present study shows that MGMT mRNA was not decreased in patients with chronic liver diseases with alcohol drinking, compared with those without alcohol drinking.

Recent reports have shown that response to interferon treatment is influenced by hepatic iron contents in patients with chronic hepatitis C. In those reports, however, hepatitis C virus (HCV) genotypes and serum HCV-RNA levels were not examined. The aim of the present study was to investigate whether hepatic iron contents influence the response to interferon in patients with chronic hepatitis C and whether HCV genotypes and serum HCV-RNA levels play a role in this relationship. Among 65 patients with chronic hepatitis C, hepatic iron contents were significantly high in patients with a history of excess drinking of alcohol (more than 80 g/day) compared to those without, and significantly low in female patients before menopause. Having excluded these patients, hepatic iron contents were significantly higher in patients with genotype 1b infection than those with genotype 2a and 2b infection. There was no significant correlation between hepatic iron contents and plasma HCV-RNA levels. Among the patients with genotype 1b infection, hepatic iron contents were significantly lower in the responders to interferon than those in the nonresponders (429 +/- 100 vs 875 +/- 110 micrograms/g liver, P < 0.05). From these results, it is concluded that response to interferon is mainly influenced by HCV genotypes, while hepatic iron contents may play an important role in response to interferon in patients with genotype 1b infection.

To characterize liver dysfunction in patients with cirrhosis after variceal bleeding, we analyzed 50 cirrhotic patients who had bleeding esophageal varices with or without shock. Increases in serum total bilirubin levels by 1.5 times were observed within 24 h in 11 of 12 patients with shock who died > 4 days after hemorrhage but in only one of eight patients with shock who survived (p < 0.01). Increases in serum aspartate aminotransferase and alanine aminotransferase by 2.5 times were observed in six patients in the former group but in none of the latter (p < 0.05). In postmortem livers, hepatocellular degeneration with minimal inflammatory cell infiltration was observed. Ischemic hepatitis is frequently noted in cirrhotic patients with ruptured esophageal varices. Patients with increases in the serum level of total bilirubin and/or aminotransferases within 24 h from onset of hemorrhage should be carefully treated even if hemorrhage is controlled.

To investigate a cellular mode of HCV-infection in the liver and its pathological implications in relation to histopathological changes or clinical data, we studied the distribution of HCV-RNA in the livers of 21 patients with HCV-related chronic liver disease (chronic active hepatitis, 14 cases; cirrhosis, 7 cases) using the in situ hybridization technique. In situ hybridization was performed on 4% paraformaldehyde-fixed frozen sections with digoxigenin-labeled DNA probe deduced from the core region of HC-J4. In situ hybridization showed positive signals in the liver specimens of 20/21 cases. The signals were localized in the cytoplasm of hepatocytes. The distribution pattern of positive cells was individually different, whereas the pattern was identical in the right and left lobes. There were no correlations of the HCV-positive cell number with serum aminotransferase levels at biopsy or with genotypes of HCV. The positive hepatocytes were occasionally associated with infiltrating mononuclear cells, and they were sparsely distributed in the area of piecemeal necrosis. These findings suggest that factors such as host immunoreaction to the virus may be more important than its direct cytopathy in the pathogenesis of chronic hepatitis C virus infection.

We analyzed 10 patients (male:female 9:1, mean age 64.2 years) who developed hepatocellular carcinoma during the observation period of 2 years among 2,164 chronic hemodialysis patients in 23 dialysis centers. Among them, 409 patients were positive for serum anti-HCV antibodies (19%). They were all positive for serum anti-HCV antibodies but negative for HBs antigen. None of the anti-HCV antibody-negative patients developed hepatocellular carcinoma during this period. Although liver function tests of the patients were almost normal, pathological examination of the liver revealed chronic active hepatitis or cirrhosis. Periodic ultrasonographic examination is necessary for early detection of hepatocellular carcinoma in chronic hemodialysis patients with positive anti-HCV antibodies even if liver function tests are within normal ranges.

Effects of cadmium on hepatic glutathione (GSH) metabolism were characterized in a human-derived hepatoma cell line, Hep G2 cells. Intracellular GSH concentrations were significantly increased after incubation with cadmium at 5 and 10 microM for 24 and 48 hr. The rate of resynthesis of GSH after depleting cellular GSH by 0.5 mM of diethylmaleate was higher in cadmium (5 microM)-pretreated cells than that in untreated controls. GSH efflux from cadmium-pretreated cells was two-fold higher than that in untreated controls. On the other hand, incubation with cadmium at 5 and 10 microM for 60 min did not decrease GSH efflux. These findings suggest that increased intracellular GSH concentrations are attributed to enhanced synthesis of GSH under cadmium exposure, although the possibility of decreased intracellular consumption of GSH should further to be studied.

We found Encephalitozoon-like organisms in an in vitro culture of a human liver lesion which was due to larval Echinococcus multilocularis. The organisms developed in the same fashion as an Encephalitozoon cuniculi. The spores that developed in parasitophorous vacuoles were 2.0-2.6 x 1.1-1.5 microns; each contained a single nucleus and 4-5 polar tubule coils, closely resembling E. cuniculi in its ultrastructure. Mature spores were collected from the supernatants by the use of Percoll centrifugation resulting in the banding of the spores on continuous gradients. We prepared three sorts of spores which were different in buoyant density in 0.15 M NaCl: 1.05-1.07 g/ml spores, 1.12 g/ml spores, and spores of over 1.14 g/ml. Polyclonal antibodies to a pool of each spore preparation were produced in a rabbit and applied to the detection of microsporidian antigen in situ. The histoimmunoperoxidase (HIP) procedure was used to detect the microsporidian antigen in echinococcal liver lesions from patients with alveolar hydatid disease (AHD). Ten echinococcal liver lesions from different AHD patients were examined and four were found to be positive in the HIP test. The Percoll-separated spores were also used as an antigen to detect for antibodies in the sera from the patients with AHD by Western blotting. Antibodies were detected in 62 (52%) of the 119 AHD patients and in only 8 (5%) of the 159 normal healthy individuals.

To study the role of antigen-specific T lymphocytes in the pathogenesis of autoimmune hepatitis, messenger RNA of T-cell receptors (TCR) was analyzed in liver biopsy specimens from four patients with autoimmune hepatitis. Using the TCR beta-chain variable region family specific oligonucleotides, a remarkable bias for the usage of beta-chain variable region 3 was detected in all four patients. Therefore, nucleotide and amino acid sequences of the complementarity-determining region 3 rearranged to the beta-chain variable region 3, which is a putative contact site for peptide fragments from antigens bound in the groove of the human leukocyte antigen molecule, was further analyzed in randomly selected 10 clones from each patient. An Asp-Arg-Pro motif in the complementarity-determining region 3 was identified in three of four patients with human leukocyte antigen DR4, and this motif was always rearranged to the beta-chain junctional region 1.2. From these results, beta-chain variable region 3+, Asp-Arg-Pro+, beta-chain junctional region 1.2+ T-cell clones may be among the responsible lymphocytes involved in the liver damage in autoimmune hepatitis, especially in patients with human leukocyte antigen DR4. Thus, an analysis of the complementarity-determining region 3 may give us an important clue to clarify characteristics of target antigens included in autoimmune hepatitis.

Nucleotide sequences of the hypervariable region of hepatitis C virus genomes obtained from plasma change rapidly during the course of infection and are believed to play a part in immunological escape and consequently in the development of persistent infection. It is not known, however, whether these changes also occur in the liver. To clarify this aspect, RNA was extracted from the plasma and liver tissue of eight patients with chronic hepatitis C. After cDNA synthesis, DNA fragments that included the hypervariable region were amplified by the polymerase chain reaction. Consensus nucleotide sequences were determined directly from the polymerase chain reaction products by the dideoxy chain termination method. The diversity of the hypervariable region was analyzed further by the polymerase chain reaction-single strand conformation polymorphism analysis. Consensus nucleotide sequences of the hypervariable region were identical between the plasma and the liver in each patient. The polymerase chain reaction-single strand conformation polymorphism analysis showed multiple DNA bands that represented different hypervariable region sequences. Comparison of the single strand conformation polymorphism patterns revealed that the number, the mobility, and the density of bands were the same between the plasma and the liver. It is concluded that the population and the diversity of hepatitis C virus quasispecies as detected by the hypervariable region sequence are the same between the plasma and the liver despite rapid mutations, indicating that rapid changes in the population of hepatitis C virus quasispecies also occur in the liver.

To study the effects of a long-term cadmium exposure on livers and kidneys, rats were administered cadmium chloride (0.228 mg Cd/kg, 3 days/week ip), for one year. Significant accumulation of cadmium was observed in livers (183 +/- 40 micrograms/g liver) and kidneys (92 +/- 17 micrograms/g kidney). Serum urea nitrogen and creatinine levels were significantly elevated in the cadmium-treated rats, while liver function tests were minimally affected. Histological observations showed interstitial fibrosis with minimal cell necrosis and inflammatory cell infiltration in livers, and apparent degeneration of proximal tubules and infiltration of inflammatory cells in parenchyma of kidneys. Lipid peroxidation in livers and kidneys, as assessed by thiobarbituric acid reactive substances, revealed no differences between the cadmium-treated rats and the controls. Glutathione contents were significantly increased in the cadmium treated rats both in livers (p < 0.001), and in kidneys (p < 0.001) compared with the controls. Increased glutathione levels in livers may contribute, in part, to the prevention of serious hepatotoxicity during chronic cadmium exposure, while nephrotoxicity due to cadmium may not be prevented by glutathione.

Effects of verapamil, an calcium channel blocker, on hepatic glutathione were studied in vivo in the rat and in the perfused rat liver. An injection of verapamil at a dose of 15 mg/kg body weight but not at 5 mg/kg significantly decreased hepatic glutathione contents in both fed and fasted animals 6 h after the injection. The administration of verapamil at a dose of 10 mg/kg twice a day for a week brought a significant decrease in hepatic glutathione contents and a significant increase in plasma glutathione levels. In the perfused rat liver, sinusoidal glutathione efflux was significantly increased when verapamil was added to the perfusion medium in a concentration of 20 microM. These data indicate that verapamil increases glutathione efflux from the liver and that calcium mobilization may be concerned in glutathione efflux in vivo.

Forty liver biopsies of hepatitis B surface antigen negative chronic alcoholics were histologically studied to assess the influence of hepatitis C virus (HCV)-infection. A moderate degree of focal hepatocellular necrosis and/or portal lymphocytic infiltration (FHN-PLI) was observed in 28% of the specimens, being especially prevalent in advanced cases of fibrosis and cirrhosis, and significantly correlated with HCV-infection. Eleven of these cases were examined in detail: HCV-infection was detected in eight and lymphocytic infiltration was apparent in the portal area, accompanied by formation of lymph follicles. The FHN-PLI was ascribed to alcoholic hepatitis in two HCV-negative cases and was concluded to be of unknown etiology in the remaining one HCV-negative case. This study thus indicated that over two-thirds of cases of chronic hepatitis in alcoholics can be attributed to HCV-infection, with the remainder being at least partly related to alcoholic hepatitis. The prevalence of alcohol-induced chronic hepatitis based on immunopathological findings was unclear, but was probably less frequent than previously reported.

To investigate the correlation between the replication of hepatitis C virus in liver and the clinical and histopathological features, we detected and quantified plus and minus strands of HCV-RNA in plasma and in livers of patients with chronic hepatitis C by a quantitative polymerase chain reaction. RNA was extracted from the plasma and liver tissue of ten patients with biopsy-proven chronic hepatitis C. The plus and minus strands of HCV-RNA were detected by a strand-specific reverse transcription with either sense or anti-sense oligonucleotide primers deduced from the hepatitis C virus genome, and a standard HCV-RNA with an enzyme restriction site was used to quantify the amount of HCV-RNA. Both plus and minus strands of HCV-RNA were detected from the liver tissue of all patients included. The amount of plus-stranded HCV-RNA in the liver was 10 times higher than that of minus-stranded HCV-RNA. Plus-stranded HCV-RNA was detected in the plasma in all patients, while the minus strand was not detected in any patient. There was a weak correlation between the amount of both strands of HCV-RNA in the liver and that of the plus strand in plasma. There was no significant correlation between the amount of liver HCV-RNA and serum alanine transaminase and aspartate transaminase levels, or histopathological findings in the liver. The present method of detecting and quantifying liver HCV-RNA is simple and sensitive; it may be used to detect residual hepatitis C virus replication after the disappearance of plasma HCV-RNA in acute hepatitis or in chronic hepatitis after interferon treatment.

Lipocytes are the major site of hepatic vitamin A storage, and they have been demonstrated to lose their vitamin A content in the process of hepatic fibrosis. To investigate the relationship between hepatic vitamin A content and the degree of hepatic fibrosis, we measured levels of retinyl palmitate and retinol in the CCl4-induced fibrotic liver using high-performance liquid chromatography. We estimated hepatic collagen content using a spectrophotometric analysis with sirius red, and also by measuring hydroxyproline levels. Lipocytes were detected by an immunoperoxidase method with anti-desmin antibody, and were counted morphometrically through a Texture Analyzing System. A significant negative correlation was observed between the level of retinyl palmitate and collagen content (r = -0.64) as well as the hydroxyproline level (r = -0.69) in the CCl4-induced fibrotic liver. In the process of fibrosis, hepatic retinol levels were elevated in association with a decrease in retinyl palmitate. In particular in the early stage of fibrosis, lipocytes increased remarkably in number in fibrotic areas in spite of a decrease in total hepatic vitamin A. The present study suggests that an increase in hepatic retinol as well as a decrease in retinyl palmitate may facilitate the process of hepatic fibrosis produced by lipocytes.

To examine HCV infection histologically, we attempted non-radioactive in situ hybridization of HCV-RNA in the liver. We amplified cDNA probe (360 base pairs) by PCR using the primers deduced from the core region of the HCV genome. The probe was labelled with digoxigenin by PCR and used for in situ hybridization on paraformaldehyde-fixed frozen liver sections. The hybrids were visualized immunohistochemically with alkaline-phosphatase-conjugated anti-digoxigenin and alkaline-phosphatase substrates. HCV-RNA-cDNA hybrids were detected in 21 of 24 patients with positive serum HCV markers, whereas there were no positive signals in the liver of 12 cases without HCV infection. The signal intensity of HCV-RNA-cDNA hybrids was abolished after RNase treatment. Various other specificity experiments also verified specific hybridization of HCV-RNA-cDNA. HCV-RNA was visualized in liver cells and most of them were regarded as hepatocytes from their characteristic features. The infected hepatocytes were frequently associated with mononuclear cell infiltration. Hepatocytes positive for HCV-RNA were sometimes binuclear and distributed in various patterns among cases tested. The present in situ hybridization of HCV RNA is highly sensitive and specific and the results suggest the host immune response to HCV-infected cells.

To determine the significance of the expression of alpha-smooth-muscle actin in the fibrotic human liver, normal and diseased livers were stained with anti-alpha-smooth-muscle-actin antibody by an immunoperoxidase method. Vitamin A-containing lipocytes were also identified by the modified Kupffer's gold chloride method. In the normal human liver, lipocytes as well as vascular smooth muscle cells expressed alpha-smooth-muscle actin. In alcoholic liver disease, there was an increase in the cells positive for alpha-smooth-muscle actin adjacent to the fibrotic areas, but the response of lipocytes to the gold chloride reaction diminished. In chronic hepatitis, the cells positive for alpha-smooth-muscle actin increased around the enlarged portal areas, and the response to the gold chloride reaction did not change appreciably. An increase in the cells positive for alpha-smooth-muscle actin was associated with the progression of hepatic fibrosis in the liver of patients with alcoholic liver disease and chronic hepatitis.

Vasopressin stimulated GSH efflux from Hep G2 cells. The maximal effect was observed at 10nM. Pretreatment with pertussis toxin or cholera toxin for 18 hr increased GSH efflux. Vasopressin-mediated GSH efflux was observed even in the cells pretreated with those compounds. Dibutyryl-cAMP or dibutyryl-cGMP enhanced GSH efflux although an additive effect of vasopressin was not observed. Glucagon and a phorbol ester independently increased GSH efflux while both compounds decreased the effect of vasopressin. Staurosporine, an inhibitor of protein kinase C, inhibited vasopressin-mediated GSH efflux. The effect of vasopressin was observed even in the absence of extracellular Ca2+. Vasopressin stimulates GSH efflux from Hep G2 cells and protein kinase C-dependent pathway may play a significant role in vasopressin-mediated GSH efflux.

To investigate the role of lymphocytes frequently observed in the parenchyma of alcoholic liver disease (ALD), lymphocytes infiltrating into the liver were stained immunohistochemically with monoclonal antibodies (MoAb) and were quantitatively assessed by a morphometric analysis in 17 patients with ALD and, for comparison in five patients with chronic active hepatitis B (B-CAH). In patients with alcoholic hepatitis, the number of CD8+ lymphocytes in the hepatic lobule was similar to that in patients with B-CAH but was significantly greater than that in alcoholics with hepatic fibrosis (HF). The CD4/CD8 ratio in the hepatic lobule was low in both alcoholic hepatitis and B-CAH compared with that of alcoholic patients with HF. When Mallory bodies (MB) and lymphocytes were simultaneously stained with a specific antibody against MB and MoAb, respectively, only CD3+ and CD8+ lymphocytes were found to have a close contact with MB. These results suggest that in alcoholic hepatitis, hepatocyte necrosis may be partly mediated by immunological mechanisms involving cytotoxic T cells infiltrating into the hepatic lobule.

Primary culture of adult rat hepatocytes resulted in marked increase of c-myc expression within a few hours. The high level of c-myc mRNA was maintained throughout culture on collagen-coated dishes, but decreased greatly with time during culture on collagen-gel or matrigel. Expression of c-myc was also down-regulated at high cell density. The decrease in its expression appeared closely related to inhibitions of DNA synthesis and cell spreading. In contrast, hepatoma H4TG cells showed a high level of c-myc expression which was not affected by culture on any extracellular matrices examined or by the cell density. These results suggest that up-regulation of expression of the c-myc gene is linked to G0 to G1 transition during cell cycle progression, which in normal hepatocytes is strictly regulated by cell-cell and cell-extracellular matrix interactions, but that this control mechanism is defective in malignant hepatic tumor cells.

Vasopression increases sinusoidal efflux of GSH in the perfused rat liver. The mechanism of this effect was studied in the perfused rat liver and in isolated rat hepatocytes. Vasopressin stimulated GSH efflux in both systems and a V1-receptor antagonist (OPC-21268) significantly inhibited the effect of vasopressin suggesting that vasopressin stimulates GSH efflux from rat hepatocytes via V1-receptor.

The presence of a-smooth muscle actin (smA)-positive cells has recently been reported in the fibrotic liver. Lipocytes have been considered to play important roles in hepatic fibrosis. However, the relation of the a-smA-positive cells and lipocytes has not been determined. The biological implication of a-smA expression remains unknown. To study these questions, we carried out double immunofluorescent staining of a-smA and desmin (a marker for lipocytes), or a-smA and collagen, and double immunohistochemical staining of a-smA and 5-bromo-2'-deoxyuridine (BrdUrd) in carbon tetrachloride-induced fibrotic rat livers. In normal and control livers, a-smA-positive cells were not seen in the lobules, whereas scattered desmin-positive cells were present. With the development of hepatic fibrosis, a-smA was expressed only in a portion of desmin-positive cells located predominantly around collagen bundles. A number of a-smA-positive cells in the lobules were labelled with BrdUrd. These results suggest phenotypic modulation in lipocytes and differentiation of lipocytes towards myofibroblast-like cells, since a-smA is expressed with desmin in myofibroblasts in scar tissue. The expression of a-smA may be related to events of the fibrotic process, such as tissue contraction or fibrogenesis per se.

The appearance of alpha-smooth-muscle-actin (alpha-smA)-positive cells during hepatic fibrosis was studied immunohistochemically in rat and human livers. In the normal rat liver, alpha-smA was observed only in vascular smooth muscle cells. With the progression of fibrosis induced by CCl4 injection, alpha-smA-positive cells appeared in the perisinusoidal space and the fibrous septa, and ultimately surrounded regenerative nodules. An increase of desmin-positive cells was recognized in the fibrotic areas and the perisinusoidal area. In the human liver, alpha-smA-positive cells appeared in the fibrotic area, whereas no desmin-positive cells were observed, except in vascular walls of the central vein and the portal tract, alpha-smA is a good marker for the detection of myofibroblast-like cells, and the appearance of alpha-smA in liver mesenchymal cells seems closely related to the process of hepatic fibrosis in both rat and man.

The existence of antibodies against alcohol-treated rabbit hepatocytes in sera of patients with alcoholic liver disease was investigated utilizing a 125I-labelled protein A assay. The sera, after two absorptions with normal rabbit hepatocytes, were incubated with hepatocytes isolated from rabbits which had been treated for 4 days with a daily dose of ethanol (1 g/kg body weight) intravenously. Serum antibodies against alcohol-treated hepatocytes were detected in 21 of 55 patients with alcoholic liver disease; this was associated with a distinct, mixed granular and linear fluorescence staining the surface of alcohol-treated hepatocytes. By contrast, none of 23 patients with other liver diseases who had no history of excessive alcohol intake showed the antibodies in their sera. The values of the antibodies of patients with alcoholic active cirrhosis and alcoholic hepatitis were significantly higher than those of other types of alcoholic liver disease. These results showed that antibodies against alcohol-treated hepatocytes are present in alcoholic liver disease with inflammation. The role of these antibodies on liver cell damage in alcoholic liver disease remains to be clarified.

The effect of replacement therapy with thyroid hormones upon RNA transcription was studied in isolated nuclei of cerebrum and liver from hypothyroid rats. The chronic administration of T4 to hypothyroid rats, from the 5th day of life, during ten days, corrected the RNA synthesis in isolated nuclei from cerebrum and liver. The injection of a single dose of T3 to neonatally thyroidectomized rats, ten days after birth, enhanced the endogeneous activity of RNA polymerase I and II of cerebrum and liver. In cerebrum the transcription of ribosomal RNA increased 40 minutes after T3 injection, this activity being constant during the whole period studied. On the other hand, the synthesis of heterogeneous RNA showed an increase only 5 hrs. after the beginning of the treatment. In liver, the lag period is higher for both enzymes, being the maximal response achieved 5 hs. after the hormonal administration.

The simultaneous administration of caffeine has been shown to potentiate acetaminophen-induced hepatotoxicity. In the present study, the mechanisms of this effect were studied. Male Sprague-Dawley rats, fasted for 18 hr, were given acetaminophen (0.5 g/kg) and/or caffeine (0.1 g/kg) i.p. Two hours later, the depletion of hepatic reduced glutathione (GSH) induced by acetaminophen was more pronounced by the concomitant administration of caffeine, although the effect of caffeine alone on the hepatic GSH content was minimal at this dose. Covalent binding of a reactive metabolite of acetaminophen to hepatic proteins was increased by caffeine although the free acetaminophen content in the liver was not affected. In isolated rat hepatocytes prepared from normal animals, caffeine enhanced acetaminophen-induced GSH depletion and potentiated covalent binding of the reactive metabolite to cellular proteins. The extracellular release of GSH + oxidized glutathione was decreased by acetaminophen, and this decrease was potentiated further by the addition of caffeine. In the cells, the production of acetaminophen-GSH conjugate also was increased in the presence of caffeine, whereas that of glucuronide conjugate was decreased. In microsomes, NADPH-dependent production of acetaminophen-GSH conjugate was increased in the presence of caffeine. Thus, caffeine appears to potentiate acetaminophen-induced hepatotoxicity mainly by enhancing the production of a reactive metabolite of acetaminophen by mixed function oxidases. To what extend caffeine-induced GSH depletion plays a role is to be clarified.

This study was undertaken to evaluate the effects of chronic ethanol consumption on the hepatotoxicity of acetaminophen. Male Sprague-Dawley rats were pair fed a nutritionally adequate liquid diet containing either ethanol or isocaloric carbohydrate for 4-6 wk. Acetaminophen (0.5 g/kg body wt) was given intraperitoneally 12 h after ethanol withdrawal. By 36 h, frank hepatic centrilobular necrosis and a decrease in hepatic aminopyrine N-demethylase activity were observed in the ethanol-fed rats, whereas in controls the changes were minimal. Serum glutamyl oxaloacetic transaminase and glutamate dehydrogenase activities were significantly increased in ethanol-fed rats. Hepatic damage in ethanol fed rats was apparent already at 6 h, as evidenced by elevated serum enzyme activities and ultrastructural changes, particularly of the mitochondria. The depletion of hepatic glutathione content and the covalent binding of acetaminophen metabolite(s) were significantly greater in ethanol-fed rats than in controls. Urinary excretion of mercapturic acid conjugate during the first 12 h was also increased in ethanol-fed rats. In an in vitro study, covalent binding of acetaminophen metabolite(s) to microsomal protein was increased after ethanol feeding for 4-6 wk. Thus, chronic ethanol feeding increases the hepatotoxicity of acetaminophen; enhanced production of reactive metabolite(s) may be responsible.